Enzymes

ABSTRACT

Various embodiments of the invention provide human enzymes (ENZM) and polynucleotides which identify and encode ENZM. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of ENZM.

TECHNICAL FIELD

The invention relates to novel nucleic acids, enzymes encoded by thesenucleic acids, and to the use of these nucleic acids and proteins in thediagnosis, treatment, and prevention of autoimmune/inflammatorydisorders, infectious disorders, immune deficiencies, disorders ofmetabolism, reproductive disorders, neurological disorders,cardiovascular disorders, eye disorders, and cell proliferativedisorders, including cancer. The invention also relates to theassessment of the effects of exogenous compounds on the expression ofnucleic acids and enzymes.

BACKGROUND OF THE INVENTION

The cellular processes of biogenesis and biodegradation involve a numberof key enzyme classes including oxidoreductases, transferases,hydrolases, lyases, isomerases, ligases, and others. Each class ofenzyme comprises many substrate-specific enzymes having precise and wellregulated functions. Enzymes facilitate metabolic processes such asglycolysis, the tricarboxylic cycle, and fatty acid metabolism;synthesis or degradation of amino acids, steroids, phospholipids, andalcohols; regulation of cell signaling, proliferation, inflammation, andapoptosis; and through catalyzing critical steps in DNA replication andrepair and the process of translation.

Oxidoreductases

Many pathways of biogenesis and biodegradation require oxidoreductase(dehydrogenase or reductase) activity, coupled to reduction or oxidationof a cofactor. Potential cofactors include cytochromes, oxygen,disulfide, iron-sulfur proteins, Ravin adenine dinucleotide (FAD), andthe nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E. A.and A. R. Leech (1983) Biochemistry for the Medical Sciences, John Wileyand Sons, Chichester, U. K. pp. 779-793). Reductase activity catalyzestransfer of electrons between substrate(s) and cofactor(s) withconcurrent oxidation of the cofactor. Reverse dehydrogenase activitycatalyzes the reduction of a cofactor and consequent oxidation of thesubstrate. Oxidoreductase enzymes are a broad superfamily that catalyzereactions in all cells of organisms, including metabolism of sugar,certain detoxification reactions, and synthesis or degradation of fattyacids, amino acids, glucocorticoids, estrogens, androgens, andprostaglandins. Different family members may be referred to asoxidoreductases, oxidases, reductases, or dehydrogenases, and they oftenhave distinct cellular locations such as the cytosol, the plasmamembrane, mitochondrial inner or outer membrane, and peroxisomes.

Short-chain alcohol dehydrogenases (SCADs) are a family ofdehydrogenases that share only 15% to 30% sequence identity, withsimilarity predominantly in the coenzyme binding domain and thesubstrate binding domain. In addition to their role in detoxification ofethanol, SCADs are involved in synthesis and degradation of fatty acids,steroids, and some prostaglandins, and are therefore implicated in avariety of disorders such as lipid storage disease, myopathy, SCADdeficiency, and certain genetic disorders. For example, retinoldehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol.Chem. 270:1107-1112) that converts retinol to retinal, the precursor ofretinoic acid. Retinoic acid, a regulator of differentiation andapoptosis, has been shown to down-regulate genes involved in cellproliferation and inflammation (Chai, X. et al. (1995) J. Biol. Chem.270:3900-3904). In addition, retinol dehydrogenase has been linked tohereditary eye diseases such as autosomal recessive childhood-onsetsevere retinal dystrophy (Simon, A. et al. (1996) Genomics 36:424-430).

Membrane-bound succinate dehydrogenases (succinate:quinone reductases,SQR) and fumarate reductases (quinol:fumarate reductases, QFR) couplethe oxidation of succinate to fumarate with the reduction of quinone toquinol, and also catalyze the reverse reaction. QFR and SQR complexesare collectively known as succinate:quinone oxidoreductases (EC 1.3.5.1)and have similar compositions. The complexes consist of two hydrophilicand one or two hydrophobic, membrane-integrated subunits. The largerhydrophilic subunit A carries covalently bound flavin adeninedinucleotide; subunit B contains three iron-sulphur centers (Lancaster,C. R. and A. Kroger (2000) Biochim. Biophys. Acta 1459:422-431). Thefull-length cDNA sequence for the flavoprotein subunit of human heartsuccinate dehydrogenase (succinate: (acceptor) oxidoreductase; EC1.3.99.1) is similar to the bovine succinate dehydrogenase in that itcontains a cysteine triplet and in that the active site contains anadditional cysteine that is not present in yeast or prokaryotic SQRs(Morris, A. A. et al. (1994) Biochim. Biophys. Acta 29:125-128).

Propagation of nerve impulses, modulation of cell proliferation anddifferentiation, induction of the immune response, and tissuehomeostasis involve neurotransmitter metabolism (Weiss, B. (1991)Neurotoxicology 12:379-386; Collins, S. M. et al. (1992) Ann. N.Y. Acad.Sci. 664:415-424; Brown, J. K. and H. Imam (1991) J. Inherit. Metab.Dis. 14:436-458). Many pathways of neurotransmitter metabolism requireoxidoreductase activity, coupled to reduction or oxidation of acofactor, such as NAD⁺/NADH (Newsholme and Leech, supra, pp. 779-793).Degradation of catecholamines (epinephrine or norepinephrine) requiresalcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (inperipheral tissue). NAD⁺-dependent aldehyde dehydrogenase oxidizes5-hydroxyindole-3-acetate (the product of 5-hydroxytryptamine(serotonin) metabolism) in the brain, blood platelets, liver andpulmonary endothelium (Newsholme and Leech, supra, p. 786). Otherneurotransmitter degradation pathways that utilize NAD⁺/NADH-dependentoxidoreductase activity include those of L-DOPA (precursor of dopamine,a neuronal excitatory compound), glycine (an inhibitory neurotransmitterin the brain and spinal cord), histamine (liberated from mast cellsduring the inflammatory response), and taurine (an inhibitoryneurotransmitter of the brain stem, spinal cord and retina) (Newsholmeand Leech, supra, pp. 790, 792). Epigenetic or genetic defects inneurotransmitter metabolic pathways can result in diseases includingParkinson disease and inherited myoclonus (McCance, K. L. and S. E.Huether (1994) Pathophysiology, Mosby-Year Book, Inc., St. Louis, Mo.pp. 402-404; Gundlach, A. L. (1990) FASEB J. 4:2761-2766).

Tetrahydrofolate is a derivatized glutamate molecule that acts as acarrier, providing activated one-carbon units to a wide variety ofbiosynthetic reactions, including synthesis of purines, pyrimidines, andthe amino acid methionine. Tetrahydrofolate is generated by the activityof a holoenzyme complex called tetrahydrofolate synthase, which includesthree enzyme activities: tetrahydrofolate dehydrogenase,tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. Thus,tetrahydrofolate dehydrogenase plays an important role in generatingbuilding blocks for nucleic and amino acids, crucial to proliferatingcells.

3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acidmetabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in themitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACDand enoyl-CoA hydratase form an enzyme complex called bifunctionalenzyme, defects in which are associated with peroxisomal bifunctionalenzyme deficiency. This interruption in fatty acid metabolism producesaccumulation of very-long chain fatty acids, disrupting development ofthe brain, bone, and adrenal glands. Infants born with this deficiencytypically die within 6 months (Watkins, P. et al. (1989) J. Clin.Invest. 83:771-777; Online Mendelian Inheritance in Man (OMIM),#261515). The neurodegeneration characteristic of Alzheimer's diseaseinvolves development of extracellular plaques in certain brain regions.A major protein component of these plaques is the peptide amyloid-β(Aβ), which is one of several cleavage products of amyloid precursorprotein (APP). 3HACD has been shown to bind the Aβ peptide, and isoverexpressed in neurons affected in Alzheimer's disease. In addition,an antibody against 3HACD can block the toxic effects of Aβ in a cellculture model of Alzheimer's disease (Yan, S. et al. (1997) Nature389:689-695; OMIM, #602057).

Steroids such as estrogen, testosterone, and corticosterone aregenerated from a common precursor, cholesterol, and interconverted.Enzymes acting upon cholesterol include dehydrogenases. Steroiddehydrogenases, such as the hydroxysteroid dehydrogenases, are involvedin hypertension, fertility, and cancer (Duax, W. L. and D. Ghosh (1997)Steroids 62:95-100). One such dehydrogenase is 3-oxo-5-α-steroiddehydrogenase (OASD), a microsomal membrane protein highly expressed inprostate and other androgen-responsive tissues. OASD catalyzes theconversion of testosterone into dihydrotestosterone, which is the mostpotent androgen. Dihydrotestosterone is essential for the formation ofthe male phenotype during embryogenesis, as well as for properandrogen-mediated growth of tissues such as the prostate and malegenitalia. A defect in OASD leads to defective formation of the externalgenitalia (Andersson, S. et al. (1991) Nature 354:159-161; Labrie, F. etal. (1992) Endocrinology 131:1571-1573; OMIM #264600).

17β-hydroxysteroid dehydrogenase (17βHSD6) plays an important role inthe regulation of the male reproductive hormone, dihydrotestosterone(DHTT). 17βHSD6 acts to reduce levels of DHTT by oxidizing a precursorof DHTT, 3α-diol, to androsterone which is readily glucuronidated andremoved. 17βHSD6 is active with both androgen and estrogen substrates inembryonic kidney 293 cells. Isozymes of 17βHSD catalyze oxidation and/orreduction reactions in various tissues with preferences for differentsteroid substrates (Biswas, M. G. and D. W. Russell (1997) J. Biol.Chem. 272:15959-15966). For example, 17βHSD1 preferentially reducesestradiol and is abundant in the ovary and placenta. 17βHSD2 catalyzesoxidation of androgens and is present in the endometrium and placenta.17βHSD3 is exclusively a reductive enzyme in the testis (Geissler, W. M.et al. (1994) Nature Genet. 7:34-39). An excess of androgens such asDHTT can contribute to diseases such as benign prostatic hyperplasia andprostate cancer.

The oxidoreductase isocitrate dehydrogenase catalyzes the conversion ofisocitrate to a-ketoglutarate, a substrate of the citric acid cycle.Isocitrate dehydrogenase can be either NAD or NADP dependent, and isfound in the cytosol, mitochondria, and peroxisomes. Activity ofisocitrate dehydrogenase is regulated developmentally, and by hormones,neurotransmitters, and growth factors.

Hydroxypyruvate reductase (HPR), a peroxisomal 2-hydroxyaciddehydrogenase in the glycolate pathway, catalyzes the conversion ofhydroxypyruvate to glycerate with the oxidation of both NADH and NADPH.The reverse dehydrogenase reaction reduces NAD⁺ and NADP⁺. HPR recyclesnucleotides and bases back into pathways leading to the synthesis of ATPand GTP, which are used to produce DNA and RNA and to control variousaspects of signal transduction and energy metabolism. Purine nucleotidebiosynthesis inhibitors are used as antiproliferative agents to treatcancer and viral diseases. HPR also regulates biochemical synthesis ofserine and cellular serine levels available for protein synthesis.

The mitochondrial electron transport (or respiratory) chain is theseries of oxidoreductase-type enzyme complexes in the mitochondrialmembrane that is responsible for the transport of electrons from NADH tooxygen and the coupling of this oxidation to the synthesis of ATP(oxidative phosphorylation). ATP provides energy to driveenergy-requiring reactions. The key respiratory chain complexes areNADH:ubiquinone oxidoreductase (complex I), succinate:ubiquinoneoxidoreductase (complex II), cytochrome c₁-b oxidoreductase (complexIII), cytochrome c oxidase (complex IV), and ATP synthase (complex V)(Alberts, B. et al. (1994) Molecular Biology of the Cell, GarlandPublishing, Inc., New York, N.Y., pp. 677-678). All of these complexesare located on the inner matrix side of the mitochondrial membraneexcept complex II, which is on the cytosolic side where it transportselectrons generated in the citric acid cycle to the respiratory chain.Electrons released in oxidation of succinate to fumarate in the citricacid cycle are transferred through electron carriers in complex II tomembrane bound ubiquinone (Q). Transcriptional regulation of thesenuclear-encoded genes controls the biogenesis of respiratory enzymes.Defects and altered expression of enzymes in the respiratory chain areassociated with a variety of disease conditions.

Other dehydrogenase activities using NAD as a cofactor include3-hydroxyisobutyrate dehydrogenase (3HBD), which catalyzes theNAD-dependent oxidation of 3-hydroxyisobutyrate to methylmalonatesemialdehyde within mitochondria. 3-hydroxyisobutyrate levels areelevated in ketoacidosis, methylmalonic acidemia, and other disorders(Rougraff, P. M. et al. (1989) J. Biol. Chem. 264:5899-5903). Anothermitochondrial dehydrogenase important in amino acid metabolism is theenzyme isovaleryl-CoA-dehydrogenase (IVD). IVD is involved in leucinemetabolism and catalyzes the oxidation of isovaleryl-CoA to3-methylcrotonyl-CoA. Human IVD is a tetrameric flavoprotein synthesizedin the cytosol with a mitochondrial import signal sequence. A mutationin the gene encoding IVD results in isovaleric acidemia (Vockley, J. etal. (1992) J. Biol. Chem. 267:2494-2501).

The family of glutathione peroxidases encompass tetrameric glutathioneperoxidases (GPx1-3) and the monomeric phospholipid hydroperoxideglutathione peroxidase (PHGPx/GPx4). Although the overall homologybetween the tetrameric enzymes and GPx4 is less than 30%, a pronouncedsimilarity has been detected in clusters involved in the active site anda common catalytic triad has been defined by structural and kinetic data(Epp, O. et al. (1983) Eur. J. Biochem. 133:51-69). GPx1 is ubiquitouslyexpressed in cells, whereas GPx2 is present in the liver and colon, andGPx3 is present in plasma. GPx4 is found at low levels in all tissuesbut is expressed at high levels in the testis (Ursini, F. et al (1995)Meth. Enzymol. 252:38-53). GPx4 is the only monomeric glutathioneperoxidase found in mammals and the only mammalian glutathioneperoxidase to show high affinity for and reactivity with phospholipidhydroperoxides, and to be membrane associated. A tandem mechanism forthe antioxidant activities of GPx4 and vitamin E has been suggested.GPx4 has alternative transcription and translation start sites whichdetermine its subcellular localization (Esworthy, R. S. et al. (1994)Gene 144:317-318; and Maiorino, M. et al. (1990) Meth. Enzymol.186:448-450).

The glutathione S-transferases (GST) are a ubiquitous family of enzymeswith dual substrate specificities that perform important biochemicalfunctions of xenobiotic biotransformation and detoxification, drugmetabolism, and protection of tissues against peroxidative damage. Theycatalyze the conjugation of an electrophile with reduced glutathione(GSH) which results in either activation or deactivation/detoxification.The absolute requirement for binding reduced GSH to a variety ofchemicals necessitates a diversity in GST structures in variousorganisms and cell types. GSTs are homodimeric or heterodimeric proteinslocalized in the cytosol. The major isozymes share common structural andcatalytic properties and include four major classes, Alpha, Mu, Pi, andTheta. Each GST possesses a common binding site for GSH, and a variablehydrophobic binding site specific for its particular electrophilicsubstrates. Specific amino acid residues within GSTs have beenidentified as important for these binding sites and for catalyticactivity. Residues Q67, T68, D101, E104, and R131 are important for thebinding of GSH (Lee, H.-C. et al. (1995) J. Biol. Chem. 270:99-109).Residues R13, R20, and R69 are important for the catalytic activity ofGST (Stenberg, G. et al. (1991) Biochem. J. 274:549-555).

GSTs normally deactivate and detoxify potentially mutagenic andcarcinogenic chemicals. Some forms of rat and human GSTs are reliablepreneoplastic markers of carcinogenesis. Dihalomethanes, which produceliver tumors in mice, are believed to be activated by GST (Thier, R. etal. (1993) Proc. Natl. Acad. Sci. USA 90:8567-8580). The mutagenicity ofethylene dibromide and ethylene dichloride is increased in bacterialcells expressing the human Alpha GST, A1-1, while the mutagenicity ofaflatoxin B1 is substantially reduced by enhancing the expression of GST(Simula, T. P. et al. (1993) Carcinogenesis 14:1371-1376). Thus, controlof GST activity may be useful in the control of mutagenesis andcarcinogenesis.

GST has been implicated in the acquired resistance of many cancers todrug treatment, the phenomenon known as multi-drug resistance (MDR). MDRoccurs when a cancer patient is treated with a cytotoxic drug such ascyclophosphamide and subsequently becomes resistant to this drug and toa variety of other cytotoxic agents as well. Increased GST levels areassociated with some drug resistant cancers, and it is believed thatthis increase occurs in response to the drug agent which is thendeactivated by the GST catalyzed GSH conjugation reaction. The increasedGST levels then protect the cancer cells from other cytotoxic agents forwhich GST has affinity. Increased levels of A1-1 in tumors has beenlinked to drug resistance induced by cyclophosphamide treatment (Dirven,H. A. et al. (1994) Cancer Res. 54:6215-6220). Thus control of GSTactivity in cancerous tissues may be useful in treating MDR in cancerpatients.

The reduction of ribonucleotides to the correspondingdeoxyribonucleotides, needed for DNA synthesis during cellproliferation, is catalyzed by the enzyme ribonucleotide diphosphatereductase. Glutaredoxin is a glutathione (GSH)-dependent hydrogen donorfor ribonucleotide diphosphate reductase and contains the active siteconsensus sequence -C-P-Y-C-. This sequence is conserved inglutaredoxins from such different organisms as Escherichia coli,vaccinia virus, yeast, plants, and mammalian cells. Glutaredoxin hasinherent GSH-disulfide oxidoreductase (thioltransferase) activity in acoupled system with GSH, NADPH, and GSH-reductase, catalyzing thereduction of low molecular weight disulfides as well as proteins.Glutaredoxin has been proposed to exert a general thiol redox control ofprotein activity by acting both as an effective protein disulfidereductase, similar to thioredoxin, and as a specific GSH-mixed disulfidereductase (Padilla, C. A. et al. (1996) FEBS Lett. 378:69-73).

In addition to their important role in DNA synthesis and cell division,glutaredoxin and other thioproteins provide effective antioxidantdefense against oxygen radicals and hydrogen peroxide (Schallreuter, K.U. and J. M. Wood (1991) Melanoma Res. 1:159-167). Glutaredoxin is theprincipal agent responsible for protein dethiolation in vivo and reducesdehydroascorbic acid in normal human neutrophils (Jung, C. H. and J. A.Thomas (1996) Arch. Biochem. Biophys. 335:61-72; Park, J. B. and M.Levine (1996) Biochem. J. 315:931-938).

The thioredoxin system serves as a hydrogen donor for ribonucleotidereductase and as a regulator of enzymes by redox control. It alsomodulates the activity of transcription factors such as NF-κB, AP-1, andsteroid receptors. Several cytokines or secreted cytokine-like factorssuch as adult T-cell leukemia-derived factor, 3B6-interleukin-1,T-hybridoma-derived (MP-6) B cell stimulatory factor, and earlypregnancy factor have been reported to be identical to thioredoxin(Holmgren, A. (1985) Annu. Rev. Biochem. 54:237-271; Abate, C. et al.(1990) Science 249:1157-1161; Tagaya, Y. et al. (1989) EMBO J.8:757-764; Wakasugi, H. (1987) Proc. Natl. Acad. Sci. USA 84:804-808;Rosen, A. et al. (1995) Int. Immunol. 7:625-633). Thus thioredoxinsecreted by stimulated lymphocytes (Yodoi, J. and T. Tursz (1991) Adv.Cancer Res. 57:381-411; Tagaya, N. et al. (1990) Proc. Natl. Acad. Sci.USA 87:8282-8286) has extracellular activities including a role as aregulator of cell growth and a mediator in the immune system(Miranda-Vizuete, A. et al. (1996) J. Biol. Chem. 271:19099-19103;Yamauchi, A. et al. (1992) Mol. Immunol. 29:263-270). Thioredoxin andthioredoxin reductase protect against cytotoxicity mediated by reactiveoxygen species in disorders such as Alzheimer's disease (Lovell, M. A.(2000) Free Radic. Biol. Med. 28:418-427).

The selenoprotein thioredoxin reductase is secreted by both normal andneoplastic cells and has been implicated as both a growth factor and asa polypeptide involved in apoptosis (Soderberg, A. et al. (2000) CancerRes. 60:2281-2289). An extracellular plasmin reductase secreted byhamster ovary cells (HT-1080) has been shown to participate in thegeneration of angiostatin from plasmin. In this case, the reduction ofthe plasmin disulfide bonds triggers the proteolytic cleavage of plasminwhich yields the angiogenesis inhibitor, angiostatin (Stathakis, P. etal. (1997) J. Biol. Chem. 272:20641-20645). Low levels of reducedsulfhydryl groups in plasma has been associated with rheumatoidarthritis. The failure of these sulfhydryl groups to scavenge activeoxygen species (e.g., hydrogen peroxide produced by activatedneutrophils) results in oxidative damage to surrounding tissues and theresulting inflammation (Hall, N. D. et al. (1994) Rheumatol. Int.4:35-38).

Another example of the importance of redox reactions in cell metabolismis the degradation of saturated and unsaturated fatty acids bymitochondrial and peroxisomal beta-oxidation enzymes which sequentiallyremove two-carbon units from Coenzyme A (CoA)-activated fatty acids. Themain beta-oxidation pathway degrades both saturated and unsaturatedfatty acids while the auxiliary pathway performs additional stepsrequired for the degradation of unsaturated fatty acids.

The pathways of rnitchondrial and peroxisomal beta-oxidation use similarenzymes, but have different substrate specificities and functions.Mitochondria oxidize short-, medium-, and long-chain fatty acids toproduce energy for cells. Mitochondrial beta-oxidation is a major energysource for cardiac and skeletal muscle. In liver, it provides ketonebodies to the peripheral circulation when glucose levels are low as instarvation, endurance exercise, and diabetes (Eaton, S. et al. (1996)Biochem. J. 320:345-357). Peroxisomes oxidize medium-, long-, andvery-long-chain fatty acids, dicarboxylic fatty acids, branched fattyacids, prostaglandins, xenobiotics, and bile acid intermediates. Thechief roles of peroxisomal beta-oxidation are to shorten toxiclipophilic carboxylic acids to facilitate their excretion and to shortenvery-long-chain fatty acids prior to mitochondrial beta-oxidation(Mannaerts, G. P. and P. P. Van Veldhoven (1993) Biochimie 75:147-158).

The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzesthe following reaction:trans-2, cis/trans-4-dienoyl-CoA+NADPH+H⁺→trans-3-enoyl-CoA+NADP⁺This reaction removes even-numbered double bonds from unsaturated fattyacids prior to their entry into the main beta-oxidation pathway(Koivuranta, K. T. et al. (1994) Biochem. J. 304:787-792). The enzymemay also remove odd-numbered double bonds from unsaturated fatty acids(Smeland, T. E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6673-6677).

Rat 2,4-dienoyl-CoA reductase is located in both mitochondria andperoxisomes (Dommes, V. et al. (1981) J. Biol. Chem. 256:8259-8262). Twoimmunologically different forms of rat mitochondrial enzyme exist withmolecular masses of 60 kDa and 120 kDa (Hakkola, E. H. and J. K.Hiltunen (1993) Eur. J. Biochem. 215:199-204). The 120 kDa mitochondrialrat enzyme is synthesized as a 335 amino acid precursor with a 29 aminoacid N-terminal leader peptide which is cleaved to form the matureenzyme (Hirose, A. et al. (1990) Biochim. Biophys. Acta 1049:346-349). Ahuman mitochondrial enzyme 83% similar to rat enzyme is synthesized as a335 amino acid residue precursor with a 19 amino acid N-terminal leaderpeptide (Koivuranta et al., supra). These cloned human and ratmitochondrial enzymes function as homotetramers (Koivuranta et al.,supra). A Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductaseis 295 amino acids long, contains a C-terminal peroxisomal targetingsignal, and functions as a homodimer (Coe, J. G. S. et al. (1994) Mol.Gen. Genet. 244:661-672; and Gurvitz, A. et al. (1997) J. Biol. Chem.272:22140-22147). All 2,4-dienoyl-CoA reductases have a fairly wellconserved NADPH binding site motif (Koivuranta et al., supra).

The main pathway beta-oxidation enzyme enoyl-CoA hydratase catalyzes thereaction:2-trans-enoyl-CoA+H₂O⇄3-hydroxyacyl-CoA

This reaction hydrates the double bond between C-2 and C-3 of2-trans-enoyl-CoA, which is generated from saturated and unsaturatedfatty acids (Engel, C. K. et al. (1996) EMBO J. 15:5135-5145). This stepis downstream from the step catalyzed by 2,4dienoyl-reductase. Differentenoyl-CoA hydratases act on short-, medium-, and long-chain fatty acids(Eaton et al., supra). Mitochondrial and peroxisomal enoyl-CoAhydratases occur as both mono-functional enzymes and as part ofmulti-functional enzyme complexes. Human liver mitochondrial short-chainenoyl-CoA hydratase is synthesized as a 290 amino acid precursor with a29 amino acid N-terminal leader peptide (Kanazawa, M. et al. (1993)Enzyme Protein 47:9-13; and Janssen, U. et al. (1997) Genomics40:470-475). Rat short-chain enoyl-CoA hydratase is 87% identical to thehuman sequence in the mature region of the protein and functions as ahomohexamer (Kanazawa et al., supra; and Engel et al., supra). Amitochondrial trifunctional protein exists that has long-chain enoyl-CoAhydratase, 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-oxothiolaseactivities (Eaton et al., supra). In human peroxisomes, enoyl-CoAhydratase activity is found in both a 327 amino acid residuemono-functional enzyme and as part of a multi-functional enzyme, alsoknown as bifunctional enzyme, which possesses enoyl-CoA hydratase,enoyl-CoA isomerase, and 3-hydroxyacyl-CoA hydrogenase activities(FitzPatrick, D. R. et al. (1995) Genomics 27:457-466; and Hoefler, G.et al. (1994) Genomics 19:60-67). A 339 amino acid residue human proteinwith short-chain enoyl-CoA hydratase activity also acts as anAU-specific RNA binding protein (Nakagawa, J. et al. (1995) Proc. Natl.Acad. Sci. USA 92:2051-2055). All enoyl-CoA hydratases share homologynear two active site glutamic acid residues, with 17 amino acid residuesthat are highly conserved (Wu, W.-J. et al. (1997) Biochemistry36:2211-2220).

Inherited deficiencies in mitochondrial and peroxisomal beta-oxidationenzymes are associated with severe diseases, some of which manifest soonafter birth and lead to death within a few years. Mitochondrialbeta-oxidation associated deficiencies include, e.g., carnitinepalmitoyl transferase and carnitine deficiency, very-long-chain acyl-CoAdehydrogenase deficiency, medium-chain acyl-CoA dehydrogenasedeficiency, short-chain acyl-CoA dehydrogenase deficiency, electrontransport flavoprotein and electron transport flavoprotein:ubiquinoneoxidoreductase deficiency, trifunctional protein deficiency, andshort-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Eaton et al.,supra). Mitochondrial trifunctional protein (including enoyl-CoAhydratase) deficient patients have reduced long-chain enoyl-CoAhydratase activities and suffer from non-ketotic hypoglycemia, suddeninfant death syndrome, cardiomyopathy, hepatic dysfunction, and muscleweakness, and may die at an early age (Eaton et al., supra).

Defects in mitochondrial beta-oxidation are associated with Reye'ssyndrome, a disease characterized by hepatic dysfunction andencephalopathy that sometimes follows viral infection in children.Reye's syndrome patients may have elevated serum levels of free fattyacids (Cotran, R. S. et al. (1994) Robbins Pathologic Basis of Disease,W.B. Saunders Co., Philadelphia Pa., p. 866). Patients withmitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency andmedium-chain 3-hydroxyacyl-CoA dehydrogenase deficiency also exhibitReye-like illnesses (Eaton et al., supra; and Egidio, R. J. et al.(1989) Am. Fam. Physician 39:221-226).

Inherited conditions associated with peroxisomal beta-oxidation includeZellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum'sdisease, acyl-CoA oxidase deficiency, peroxisomal thiolase deficiency,and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J.Hum. Genet. 54:36-43; Hoefler et al., supra). Patients with peroxisomalbifunctional enzyme deficiency, including that of enoyl-CoA hydratase,suffer from hypotonia, seizures, psychomotor defects, and defectiveneuronal migration; accumulate very-long-chain fatty acids; andtypically die within a few years of birth (Watkins, P. A. et al. (1989)J. Clin. Invest. 83:771-777).

Peroxisomal beta-oxidation is impaired in cancerous tissue. Althoughneoplastic human breast epithelial cells have the same number ofperoxisomes as do normal cells, fatty acyl-CoA oxidase activity is lowerthan in control tissue (el Bouhtoury, F. et al. (1992) J. Pathol.166:27-35). Human colon carcinomas have fewer peroxisomes than normalcolon tissue and have lower fatty-acyl-CoA oxidase and bifunctionalenzyme (including enoyl-CoA hydratase) activities than normal tissue(Cable, S. et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol.Pathol. 62:221-226).

6-phosphogluconate dehydrogenase (6-PGDH) catalyses the NADP⁺-dependentoxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphatewith the production of NADPH. The absence or inhibition of 6-PGDHresults in the accumulation of 6-phosphogluconate to toxic levels ineukaryotic cells. 6-PGDH is the third enzyme of the pentose phosphatepathway (PPP) and is ubiquitous in nature. In some heterofermentatativespecies, NAD+ is used as a cofactor with the subsequent production ofNADH.

The reaction proceeds through a 3-keto intermediate which isdecarboxylated to give the enol of ribulose 5-phosphate, then convertedto the keto product following tautomerization of the enol (Berdis A. J.and P. F. Cook (1993) Biochemistry 32:2041-2046). 6-PGDH activity isregulated by the inhibitory effect of NADPH, and the activating effectof 6-phosphogluconate (Rippa, M. et al. (1998) Biochim. Biophys. Acta1429:83-92). Deficiencies in 6-PGDH activity have been linked to chronichemolytic anemia.

The targeting of specific forms of 6-PGDH (e.g., enzymes found intrypanosomes) has been suggested as a means for controlling parasiticinfections (Tetaud, E. et al. (1999) Biochem. J. 338:55-60). Forexample, the Trypanosoma brucei enzyme is markedly more sensitive toinhibition by the substrate analogue 6-phospho-2-deoxygluconate and thecoenzyme analogue adenosine 2′,5′-bisphosphate, compared to themammalian enzyme (Hanau, S. et al. (1996) Eur. J. Biochem. 240:592-599).

Ribonucleotide diphosphate reductase catalyzes the reduction ofribonucleotide diphosphates (i.e., ADP, GDP, CDP, and UDP) to theircorresponding deoxyribonucleotide diphosphates (i.e., dADP, dGDP, dCDP,and dUDP) which are used for the synthesis of DNA. Ribonucleotidediphosphate reductase thereby performs a crucial role in the de novosynthesis of deoxynucleotide precursors. Deoxynucleotides are alsoproduced from deoxynucleosides by nucleoside kinases via the salvagepathway.

Mammalian ribonucleotide diphosphate reductase comprises two components,an effector-binding component (E) and a non-heme iron component (F).Component E binds the nucleoside triphosphate effectors while componentF contains the iron radical necessary for catalysis. Molecular weightdeterminations of the E and F components, as well as the holoenzyme,vary according to the methods used in purification of the proteins andthe particular laboratory. Component E is approximately 90-100 kDa,component F is approximately 100-120 kDa, and the holoenzyme is 200-250kDa.

Ribonucleotide diphosphate reductase activity is adversely effected byiron chelators, such as thiosemicarbazones, as well as EDTA.Deoxyribonucleotide diphosphates also appear to be negative allostericeffectors of ribonucleotide diphosphate reductase. Nucleotidetriphosphates (both ribo- and deoxyribo-) appear to stimulate theactivity of the enzyme. 3-methyl-4-nitrophenol, a metabolite of widelyused organophosphate pesticides, is a potent inhibitor of ribonucleotidediphosphate reductase in mammalian cells. Some evidence suggests thatribonucleotide diphosphate reductase activity in DNA virus (e.g., herpesvirus)-infected cells and in cancer cells is less sensitive toregulation by allosteric regulators and a correlation exists betweenhigh ribonucleotide diphosphate reductase activity levels and high ratesof cell proliferation (e.g., in hepatomas). This observation suggeststhat virus-encoded ribonucleotide diphosphate reductases, and thosepresent in cancer cells, are capable of maintaining an increased supplydeoxyribonucleotide pool for the production of virus genomes or for theincreased DNA synthesis which characterizes cancers cells.Ribonucleotide diphosphate reductase is thus a target for therapeuticintervention (Nutter, L. M. and Y.-C. Cheng (1984) Pharmac. Ther.26:191-207; and Wright, J. A. (1983) Pharmac. Ther. 22:81-102).

Dihydrodiol dehydrogenases (DD) are monomeric, NAD(P)⁺-dependent, 34-37kDa enzymes responsible for the detoxification of trans-dihydrodiol andanti-diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH)such as benzo[α]yrene, benz[α]anthracene, 7-methyl-benz[α]anthracene,7,12-dimethyl-benz[α]anthracene, chrysene, and 5-methyl-chrysene. Inmammalian cells, an environmental PAH toxin such as benzo[α]yrene isinitially epoxidated by a microsomal cytochrome P450 to yield7R,8R-arene-oxide and subsequently (−)-7R,8R-dihydrodiol((−)-trans-7,8-dihydroxy-7,8-dihydrobenzo[α]pyrene or(−)-trans-B[α]P-diol) This latter compound is further transformed to theanti-diol epoxide of benzo[α]pyrene (i.e.,(±)-anti-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzol[α]pyrene),by the same enzyme or a different enzyme, depending on the species. Thisresulting anti-diol epoxide of benzo[α]yrene, or the correspondingderivative from another PAH compound, is highly mutagenic.

DD efficiently oxidizes the precursor of the anti-diol epoxide (i.e.,trans-dihydrodiol) to transient catechols which auto-oxidize toquinones, also producing hydrogen peroxide and semiquinone radicals.This reaction prevents the formation of the highly carcinogenicanti-diol. Anti-diols are not themselves substrates for DD yet theaddition of DD to a sample comprising an anti-diol compound results in asignificant decrease in the induced mutation rate observed in the Amestest. In this instance, DD is able to bind to and sequester theanti-diol, even though it is not oxidized. Whether through oxidation orsequestration, DD plays an important role in the detoxification ofmetabolites of xenobiotic polycyclic compounds (Penning, T. M. (1993)Chemico-Biological Interactions 89:1-34).

15-oxoprostaglandin 13-reductase (PGR) and 15-hydroxyprostaglandindehydrogenase (15-PGDH) are enzymes present in the lung that areresponsible for degrading circulating prostaglandins. Oxidativecatabolism via passage through the pulmonary system is a common means ofreducing the concentration of circulating prostaglandins. 15-PGDHoxidizes the 15-hydroxyl group of a variety of prostaglandins to producethe corresponding 15-oxo compounds. The 15-oxo derivatives usually havereduced biological activity compared to the 15-hydroxyl molecule. PGRfurther reduces the 13,14 double bond of the 15-oxo compound whichtypically leads to a further decrease in biological activity. PGR is amonomer with a molecular weight of approximately 36 kDa. The enzymerequires NADH or NADPH as a cofactor with a preference for NADH. The15-oxo derivatives of prostaglandins PGE₁, PGE₂, and PGE_(2α), are allsubstrates for PGR; however, the non-derivatized prostaglandins (i.e.,PGE₁, PG₂, and PGE_(2α)) are not substrates (Ensor, C. M. et al. (1998)Biochem. J. 330:103-108).

15-PGDH and PGR also catalyze the metabolism of lipoxin A₄ (LXA₄).Lipoxins (LX) are autacoids, lipids produced at the sites of localizedinflammation, which down-regulate polymorphonuclear leukocyte (PMN)function and promote resolution of localized trauma. Lipoxin productionis stimulated by the administration of aspirin in that cells displayingcyclooxygenase II (COX II) that has been acetylated by aspirin and cellsthat possess 5-lipoxygenase (5-LO) interact and produce lipoxin. 15-PGDHgenerates 15-oxo-LXA₄ with PGR further converting the 15-oxo compound to13,14-dihydro-15-oxo-LXA₄ (Clish, C. B. et al. (2000) J. Biol. Chem.275:25372-25380). This finding suggests a broad substrate specificity ofthe prostaglandin dehydrogenases and has implications for these enzymesin drug metabolism and as targets for therapeutic intervention toregulate inflammation.

The GMC (glucose-methanol-choline) oxidoreductase family of enzymes wasdefined based on sequence alignments of Drosophila melanogaster glucosedehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus nigerglucose oxidase, and Hansenula polymorpha methanol oxidase. Despitetheir different sources and substrate specificities, these fourflavoproteins are homologous, being characterized by the presence ofseveral distinctive sequence and structural features. Each moleculecontains a canonical ADP-binding, beta-alpha-beta mononucleotide-bindingmotif close to the amino terminus. This fold comprises a four-strandedparallel beta-sheet sandwiched between a three-stranded antiparallelbeta-sheet and alpha-helices. Nucleotides bind in similar positionsrelative to this chain fold (Cavener, D. R. (1992) J. Mol. Biol.223:811-814; Wierenga, R. K. et al. (1986) J. Mol. Biol. 187:101-107).Members of the GMC oxidoreductase family also share a consensus sequencenear the central region of the polypeptide. Additional members of theGMC oxidoreductase family include cholesterol oxidases fromBrevibacterium sterolicum and Streptomyces; and an alcohol dehydrogenasefrom Pseudomonas oleovorans (Cavener, supra; Henikoff, S. and J. G.Henikoff (1994) Genomics 19:97-107; van Beilen, J. B. et al. (1992) Mol.Microbiol. 6:3121-3136).

IMP dehydrogenase and GMP reductase are two oxidoreductases which sharemany regions of sequence similarity. IMP dehydrogenase (EC 1.1.1.205)catalyes the NAD-dependent reduction of IMP (inosine monophosphate) intoXMP (xanthine monophosphate) as part of de novo GTP biosynthesis(Collart, F. R. and E. Huberman (1988) J. Biol. Chem. 263:15769-15772).GMP reductase catalyzes the NADPH-dependent reductive deamination of GMPinto IMP, helping to maintain the intracellular balance of adenine andguanine nucleotides (Andrews, S. C. and J. R. Guest (1988) Biochem. J.255:35-43).

Pyridine nucleotide-disulphide oxidoreductases are FAD flavoproteinsinvolved in the transfer of reducing equivalents from FAD to asubstrate. These flavoproteins contain a pair of redox-active cysteinescontained within a consensus sequence which is characteristic of thisprotein family (Kurlyan, J. et al. (1991) Nature 352:172-174). Membersof this family of oxidoreductases include glutathione reductase (C1.6.4.2); thioredoxin reductase of higher eukaryotes (EC 1.6.4.5);trypanothione reductase (EC 1.6.4.8); lipoamide dehydrogenase (EC1.8.1.4), the E3 component of alpha-ketoacid dehydrogenase complexes;and mercuric reductase (EC 1.16.1.1).

Transferases

Transferases are enzymes that catalyze the transfer of molecular groups.The reaction may involve an oxidation, reduction, or cleavage ofcovalent bonds, and is often specific to a substrate or to particularsites on a type of substrate. Transferases participate in reactionsessential to such functions as synthesis and degradation of cellcomponents, and regulation of cell functions including cell signaling,cell proliferation, inflammation, apoptosis, secretion and excretion.Transferases are involved in key steps in disease processes involvingthese functions. Transferases are frequently classified according to thetype of group transferred. For example, methyl transferases transferone-carbon methyl groups, amino transferases transfer nitrogenous aminogroups, and similarly denominated enzymes transfer aldehyde or ketone,acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl,phosphorous-containing, sulfur-containing, or selenium-containinggroups, as well as small enzymatic groups such as Coenzyme A.

Acyl transferases include peroxisomal carnitine octanoyl transferase,which is involved in the fatty acid beta-oxidation pathway, andmitochondrial carnitine palmitoyl transferases, involved in fatty acidmetabolism and transport. Choline O-acetyl transferase catalyzes thebiosynthesis of the neurotransmitter acetylcholine. N-acyltransferaseenzymes catalyze the transfer of an amino acid conjugate to an activatedcarboxylic group. Endogenous compounds and xenobiotics are activated byacyl-CoA synthetases in the cytosol, microsomes, and mitochondria. Theacyl-CoA intermediates are then conjugated with an amino acid (typicallyglycine, glutamine, or taurine, but also ornithine, arginine, histidine,serine, aspartic acid, and several dipeptides) by N-acyltransferases inthe cytosol or mitochondria to form a metabolite with an amide bond. Onewell-characterized enzyme of this class is the bile acid-CoA:amino acidN-acyltransferase (BAT) responsible for generating the bile acidconjugates which serve as detergents in the gastrointestinal tract(Falany, C. N. et al. (1994) J. Biol. Chem. 269:19375-19379; Johnson, M.R. et al. (1991) J. Biol. Chem. 266:10227-10233). BAT is also useful asa predictive indicator for prognosis of hepatocellular carcinomapatients after partial hepatectomy (Furutani, M. et al. (1996)Hepatology 24:1441-1445).

Acetyltransferases

Acetyltransferases have been extensively studied for their role inhistone acetylation. Histone acetylation results in the relaxing of thechromatin structure in eukaryotic cells, allowing transcription factorsto gain access to promoter elements of the DNA templates in the affectedregion of the genome (or the genome in general). In contrast, histonedeacetylation results in a reduction in transcription by closing thechromatin structure and limiting access of transcription factors. Tothis end, a common means of stimulating cell transcription is the use ofchemical agents that inhibit the deacetylation of histones (e.g., sodiumbutyrate), resulting in a global (albeit artifactual) increase in geneexpression. The modulation of gene expression by acetylation alsoresults from the acetylation of other proteins, including but notlimited to, p53, GATA-1, MyoD, ACTR, TFIIE, TFIIF and the high mobilitygroup proteins (HMG). In the case of p53, acetylation results inincreased DNA binding, leading to the stimulation of transcription ofgenes regulated by p53. The prototypic histone acetylase (HAT) is Gcn5from Saccharomyces cerevisiae. Gcn5 is a member of a family ofacetylases that includes Tetrahymena p55, human Gcn5, and humanp300/CBP. Histone acetylation is reviewed in (Cheung, W. L. et al.(2000) Curr. Opin. Cell Biol. 12:326-333 and Berger, S. L. (1999) Curr.Opin. Cell Biol. 11:336-341). Some acetyltransferase enzymes possess thealpha/beta hydrolase fold (Center of Applied Molecular Engineering Inst.of Chemistry and Biochemistry—University of Salzburg,http://predict.sanger.ac.uk/irbm-course97/Docs/ms/) common to severalother major classes of enzymes, including but not limited to,acetylcholinesterases and carboxylesterases (Structural Classificationof Proteins, http:flscop.mrc-1mb.cam.ac.uk/scop/index.html).

N-acetyltransferases are cytosolic enzymes which utilize the cofactoracetyl-coenzyme A (acetyl-CoA) to transfer the acetyl group to aromaticamines and hydrazine containing compounds. In humans, there are twohighly similar N-acetyltransferase enzymes, NAT1 and NAT2; mice appearto have a third form of the enzyme, NAT3. The human forms ofN-acetyltransferase have independent regulation (NAT1 iswidely-expressed, whereas NAT2 is in liver and gut only) and overlappingsubstrate preferences. Both enzymes appear to accept most substrates tosome extent, but NAT1 does prefer some substrates (para-aminobenzoicacid, para-aminosalicylic acid, sulfamethoxazole, and sulfanilamide),while NAT2 prefers others (isoniazid, hydralazine, procainamide,dapsone, aminoglutethimide, and sulfamethazine). A recently isolatedhuman gene, tubedown-1, is homologous to the yeast NAT-1N-acetyltransferases and encodes a protein associated withacetyltransferase activity. The expression patterns of tubedown-1suggest that it may be involved in regulating vascular and hematopoieticdevelopment (Gendron, R. L. et al. (2000) Dev. Dyn. 218:300-315).

Amino transferases comprise a family of pyridoxal 5′-phosphate(PLP)-dependent enzymes that catalyze transformations of amino acids.Amino transferases play key roles in protein synthesis and degradation,and they contribute to other processes as well. For example, GABAaminotransferase (GABA-T) catalyzes the degradation of GABA, the majorinhibitory amino acid neurotransmitter. The activity of GABA-T iscorrelated to neuropsychiatric disorders such as alcoholism, epilepsy,and Alzheimer's disease (Sherif, F. M. and S. S. Ahmed (1995) Clin.Biochem. 28:145-154). Other members of the family include pyruvateaminotransferase, branched-chain amino acid aminotransferase, tyrosineaminotransferase, aromatic aminotransferase, alanine:glyoxylateaminotransferase (AGT), and kynurenine aminotransferase (Vacca, R. A. etal. (1997) J. Biol. Chem. 272:21932-21937). Kynurenine aminotransferasecatalyzes the irreversible transamination of the L-tryptophan metaboliteL-kynurenine to form kynurenic acid. The enzyme may also catalyzes thereversible transamination reaction between L-2-aminoadipate and2-oxoglutarate to produce 2-oxoadipate and L-glutamate. Kynurenic acidis a putative modulator of glutamatergic neurotransmission, thus adeficiency in kynurenine aminotransferase may be associated withpleiotropic effects (Buchli, R. et al. (1995) J. Biol. Chem.270:29330-29335).

Glycosyl transferases include the mammalian UDP-glucouronosyltransferases, a family of membrane-bound microsomal enzymes catalyzingthe transfer of glucouronic acid to lipophilic substrates in reactionsthat play important roles in detoxification and excretion of drugs,carcinogens, and other foreign substances. Another mammalian glycosyltransferase, mammalian UDP-galactose-ceramide galactosyl transferase,catalyzes the transfer of galactose to ceramide in the synthesis ofgalactocerebrosides in myelin membranes of the nervous system. TheUDP-glycosyl transferases share a conserved signature domain of about 50amino acid residues (PROSITE: PDOC00359,http://expasy.hcuge.ch/sprot/prosite.html).

Methyl transferases are involved in a variety of pharmacologicallyimportant processes. Nicotinamide N-methyl transferase catalyzes theN-methylation of nicotinamides and other pyridines, an important step inthe cellular handling of drugs and other foreign compounds.Phenylethanolamine N-methyl transferase catalyzes the conversion ofnoradrenalin to adrenalin. 6-O-methylguanine-DNA methyl transferasereverses DNA methylation, an important step in carcinogenesis.Uroporphyrin-III C-methyl transferase, which catalyzes the transfer oftwo methyl groups from S-adenosyl-L-methionine to uroporphyrinogen III,is the first specific enzyme in the biosynthesis of cobalamin, a dietaryenzyme whose uptake is deficient in pernicious anemia. Protein-argininemethyl transferases catalyze the posttranslational methylation ofarginine residues in proteins, resulting in the mono- and dimethylationof arginine on the guanidino group. Substrates include histones, myelinbasic protein, and heterogeneous nuclear ribonucleoproteins involved inmRNA processing, splicing, and transport. Protein-arginine methyltransferase interacts with proteins upregulated by mitogens, withproteins involved in chronic lymphocytic leukemia, and with interferon,suggesting an important role for methylation in cytokine receptorsignaling (Lin, W.-J. et al. (1996) J. Biol. Chem. 271:15034-15044;Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H. S. et al.(1998) Genomics 48:330-340).

Phospho transferases catalyze the transfer of high-energy phosphategroups and are important in energy-requiring and -releasing reactions.The metabolic enzyme creatine kinase catalyzes the reversible phosphatetransfer between creatine/creatine phosphate and ATP/ADP. Glycocyaminekinase catalyzes phosphate transfer from ATP to guanidoacetate, andarginine kinase catalyzes phosphate transfer from ATP to arginine. Acysteine-containing active site is conserved in this family (PROSITE:PDOC00103).

Prenyl transferases are heterodimers, consisting of an alpha and a betasubunit, that catalyze the transfer of an isoprenyl group. The Rasfarnesyltransferase (FTase) enzyme transfers a farnesyl moiety fromcytosolic farnesylpyrophosphate to a cysteine residue at the carboxylterminus of the Ras oncogene protein. This modification is required toanchor Ras to the cell membrane so that it can perform its role insignal transduction. FTase inhibitors block Ras function and demonstrateantitumor activity (Buolamwini, J. K. (1999) Curr. Opin. Chem. Biol.3:500-509). Ftase, which shares structural similarity withgeranylgeranyl transferase, or Rab GG transferase, prenylates Rabproteins, allowing them to perform their roles in regulating vesicletransport (Seabra, M. C. (1996) J. Biol. Chem. 271:14398-14404).

Saccharyl transferases are glycating enzymes involved in a variety ofmetabolic processes. Oligosaccharyl transferase-48, for example, is areceptor for advanced glycation endproducts, which accumulate invascular complications of diabetes, macrovascular disease, renalinsufficiency, and Alzheimer's disease (Thornalley, P. J. (1998) CellMol. Biol. (Noisy-Le-Grand) 44:1013-1023).

Coenzyme A (CoA) transferase catalyzes the transfer of CoA between twocarboxylic acids. Succinyl CoA:3-oxoacid CoA transferase, for example,transfers CoA from succinyl-CoA to a recipient such as acetoacetate.Acetoacetate is essential to the metabolism of ketone bodies, whichaccumulate in tissues affected by metabolic disorders such as diabetes(PROSITE: PDOC00980).

Transglutaminase transferases (Tgases) are Ca²⁺ dependent enzymescapable of forming isopeptide bonds by catalyzing the transfer of theγ-carboxy group from protein-bound glutamine to the ε-amino group ofprotein-bound lysine residues or other primary amines. Tgases are theenzymes responsible for the cross-lining of cornified envelope (CE), thehighly insoluble protein structure on the surface of corneocytes, into achemically and mechanically resistant protein polymer. Seven known humanTgases have been identified. Individual transglutaminase gene productsare specialized in the cross-linking of specific proteins or tissuestructures, such as factor XIIIa which stabilizes the fibrin clot inhemostasis, prostrate transglutaminase which functions in semencoagulation, and tissue transglutaminase which is involved inGTP-binding in receptor signaling. Four (Tgases 1, 2, 3, and X) areexpressed in terminally differentiating epithelia such as the epidermis.Tgases are critical for the proper cross-inking of the CE as seen in thepathology of patients suffering from one form of the skin diseasesreferred to as congenital ichthyosis which has been linked to mutationsin the keratinocyte transglutaminase (TG_(K)) gene (Nemes, Z. et al.(1999) Proc. Natl. Acad. Sci. U.S.A. 96:8402-8407, Aeschlimann, D. etal. (1998) J. Biol. Chem. 273:3452-3460.)

Hydrolases

Hydrolases are a class of enzymes that catalyze the cleavage of variouscovalent bonds in a substrate by the introduction of a molecule ofwater. The reaction involves a nucleophilic attack by the watermolecule's oxygen atom on a target bond in the substrate. The watermolecule is split across the target bond, breaking the bond andgenerating two product molecules. Hydrolases participate in reactionsessential to such functions as synthesis and degradation of cellcomponents, and for regulation of cell functions including cellsignaling, cell proliferation, inflammation, apoptosis, secretion andexcretion. Hydrolases are involved in key steps in disease processesinvolving these functions. Hydrolytic enzymes, or hydrolases, may begrouped by substrate specificity into classes including phosphatases,peptidases, lysophospholipases, phosphodiesterases, glycosidases,glyoxalases, aminohydrolases, carboxylesterases, sulfatases,phosphohydrolases, nucleotidases, lysozymes, and many others.

Phosphatases hydrolytically remove phosphate groups from proteins, anenergy-providing step that regulates many cellular processes, includingintracellular signaling pathways that in turn control cell growth anddifferentiation, cell-cell contact, the cell cycle, and oncogenesis.

Peptidases, also called proteases, cleave peptide bonds that form thebackbone of peptide or protein chains. Proteolytic processing isessential to cell growth, differentiation, remodeling, and homeostasisas well as inflammation and the immune response. Since typical proteinhalf-lives range from hours to a few days, peptidases are continuallycleaving precursor proteins to their active form, removing signalsequences from targeted proteins, and degrading aged or defectiveproteins. Peptidases function in bacterial, parasitic, and viralinvasion and replication within a host. Examples of peptidases includetrypsin and chymotrypsin (components of the complement cascade and theblood-clotting cascade) lysosomal cathepsins, calpains, pepsin, renin,and chymosin (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: APractical Approach, Oxford University Press, New York, N.Y., pp. 1-5).

Lysophospholipases (LPLs) regulate intracellular lipids by catalyzingthe hydrolysis of ester bonds to remove an acyl group, a key step inlipid degradation. Small LPL isoforms, approximately 15-30 kD, functionas hydrolases; larger isoforms function both as hydrolases andtransacylases. A particular substrate for LPLs, lysophosphatidylcholine,causes lysis of cell membranes. LPL activity is regulated by signalingmolecules important in numerous pathways, including the inflammatoryresponse.

The phosphodiesterases catalyze the hydrolysis of one of the two esterbonds in a phosphodiester compound. Phosphodiesterases are thereforecrucial to a variety of cellular processes. Phosphodiesterases includeDNA and RNA endo- and exo-nucleases, which are essential to cell growthand replication as well as protein synthesis. Endonuclease V(deoxyinosine 3′-endonuclease) is an example of a type II site-specificdeoxyribonuclease, a putative DNA repair enzyme that cleaves DNAscontaining hypoxanthine, uracil, or mismatched bases. Escherichia coliendonuclease V has been shown to cleave DNA containing deoxyxanthosineat the second phosphodiester bond 3′ to deoxyxanthosine, generating a3′-hydroxyl and a 5′-phosphoryl group at the nick site (He, B. et al.(2000) Mutat. Res. 459:109-114). It has been suggested that Escherichiacoli endonuclease V plays a role in the removal of deaminated guanine,i.e., xanthine, from DNA, thus helping to protect the cell against themutagenic effects of nitrosative deamination (Schouten, K. A. and B.Weiss (1999) Mutat. Res. 435:245-254). In eukaryotes, the process oftRNA splicing requires the removal of small tRNA introns that interruptthe anticodon loop 1 base 3′ to the anticodon. This process requires thestepwise action of an endonuclease, a ligase, and a phosphotransferase(Hong, L. et al. (1998) Science 280:279-284). Ribonuclease P (RNase P)is a ubiquitous RNA processing endonuclease that is required forgenerating the mature tRNA 5′-end during the tRNA splicing process. Thisis accomplished through the catalysis of the cleavage of P-3′O bonds toproduce 5′-phosphate and 3′-hydroxyl end groups at a specific site onpre-tRNA. Catalysis by RNase P is absolutely dependent on divalentcations such as Mg²⁺ or Mn²⁺ (Kurz, J. C. et al. (2000) Curr. Opin.Chem. Biol. 4:553-558). Substrate recognition mechanisms of RNase P arewell conserved among eukaryotes and bacteria (FENZMi, S. et al. (1998)Science 280:284-286). In Saccharomyces cerevisiae, POP1 (‘processing ofprecursor RNAs’) encodes a protein component of both RNase P and RNaseMRP, another RNA processing protein. Mutations in yeast POP1 are lethal(Lygerou, Z. et al. (1994) Genes Dev. 8:1423-1433). Anotherphosphodiesterase, acid sphingomyelinase, hydrolyzes the membranephospholipid sphingomyelin to ceramide and phosphorylcholine.Phosphorylcholine functions in synthesis of phosphatidylcholine, whichis involved in intracellular signaling pathways. Ceramide is anessential precursor for the generation of gangliosides, membrane lipidsfound in high concentration in neural tissue. Defective acidsphingomyelinase phosphodiesterase leads to Niemann-Pick disease.

Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides,which are compounds that contain one or more sugar. Mammalianlactase-phlorizin hydrolase, for example, is an intestinal enzyme thatsplits lactose. Mammalian beta-galactosidase removes the terminalgalactose from gangliosides, glycoproteins, and glycosaminoglycans, anddeficiency of this enzyme is associated with a gangliosidosis known asMorquio disease type B (PROSITE PCDOC00910). Vertebrate lysosomalalpha-glucosidase, which hydrolyzes glycogen, maltose, and isomaltose,and vertebrate intestinal sucrase-isomaltase, which hydrolyzes sucrose,maltose, and isomaltose, are widely distributed members of this familywith highly conserved sequences at their active sites.

The glyoxylase system is involved in gluconeogenesis, the production ofglucose from storage compounds in the body. It consists of glyoxylase I,which catalyzes the formation of S-D-lactoylglutathione frommethyglyoxal, a side product of triose-phosphate energy metabolism, andglyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acidand reduced glutathione. Glyoxylases are involved in hyperglycemia,non-insulin-dependent diabetes mellitus, the detoxification of bacterialtoxins, and in the control of cell proliferation and microtubuleassembly.

NG,NG-dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme thathydrolyzes the endogenous nitric oxide synthase (NOS) inhibitors,NG-monomethyl-arginine and NG,NG-dimethyl-L-arginine, to L-citrulline.Inhibiting DDAH can cause increased intracellular concentration of NOSinhibitors to levels sufficient to inhibit NOS. Therefore, DDAHinhibition may provide a method of NOS inhibition, and changes in theactivity of DDAH could play a role in pathophysiological alterations innitric oxide generation (MacAllister, R. J. et al. (1996) Br. J.Pharmacol. 119:1533-1540). DDAH was found in neurons displayingcytoskeletal abnormalities and oxidative stress in Alzheimer's disease.In age-matched control cases, DDAH was not found in neurons. Thissuggests that oxidative stress- and nitric oxide-mediated events play arole in the pathogenesis of Alzheimer's disease (Smith, M. A. et al.(1998) Free Rad. Biol. Med. 25:898-902).

Acyl-CoA thioesterase is another member of the carboxylesterase family(Alexson, S. E. et al. (1993) Eur. J. Biochem. 214:719-727). Evidencesuggests that acyl-CoA thioesterase has a regulatory role insteroidogenic tissues (Finkielstein, C. et al. (1998) Eur. J. Biochem.256:60-66).

The alpha/beta hydrolase protein fold is common to several hydrolases ofdiverse phylogenetic origin and catalytic function. Enzymes with thealpha/beta hydrolase fold have a common core structure consisting ofeight beta-sheets connected by alpha-helices. The most conservedstructural feature of this fold is the loops of thenucleophile-histidine-acid catalytic triad. The histidine in thecatalytic triad is completely conserved, while the nucleophile and acidloops accommodate more than one type of amino acid (Ollis, D. L. et al.(1992) Protein Eng. 5:197-211).

Sulfatases are members of a highly conserved gene family that shareextensive sequence homology and a high degree of structural similarity.Sulfatases catalyze the cleavage of sulfate esters. To perform thisfunction, sulfatases undergo a unique post-translational modification inthe endoplasmic reticulum that involves the oxidation of a conservedcysteine residue. A human disorder called multiple sulfatase deficiencyis due to a defect in this post-translational modification step, leadingto inactive sulfatases (Recksiek, M. et al. (1998) J. Biol. Chem.273:6096-6103).

Phosphohydrolases are enzymes that hydrolyze phosphate esters. Somephosphohydrolases contain a mutT domain signature sequence. MutT is aprotein involved in the GO system responsible for removing anoxidatively damaged form of guanine from DNA. A region of about 40 aminoacid residues, found in the N-terminus of mutT, is also found in otherproteins, including some phosphohydrolases (PROSITE PDOC00695).

Serine hydrolases are a large functional class of hydrolytic enzymesthat contain a serine residue in their active site. This class ofenzymes contains proteinases, esterases, and lipases which hydrolyze avariety of substrates and, therefore, have different biological roles.Proteins in this superfamily can be further grouped into subfamiliesbased on substrate specificity or amino acid similarities (Puente, X. S.and C. Lopez-Otin (1995) J. Biol. Chem. 270:12926-12932).

Neuropathy target esterase (NTE) is an integral membrane protein presentin all neurons and in some non-neural-cell types of vertebrates. NTE isinvolved in a cell-signaling pathway controlling interactions betweenneurons and accessory glial cells in the developing nervous system. NTEhas serine esterase activity and efficiently catalyses the hydrolysis ofphenyl valerate (PV) in vitro, but its physiological substrate isunknown. NTE is not related to either the major serine esterase family,which includes acetylcholinesterase, nor to any other known serinehydrolases. NTE contains at least two functional domains: an N-terminalputative regulatory domain and a C-terminal effector domain whichcontains the esterase activity and is, in part, conserved in proteinsfound in bacteria, yeast, nematodes and insects. NTE's effector domaincontains three predicted transmembrane segments, and the active-siteserine residue lies at the center of one of these segments. The isolatedrecombinant domain shows PV hydrolase activity only when incorporatedinto phospholipid liposomes. NTE's esterase activity is largelyredundant in adult vertebrates, but organophosphates which react withNTE in vivo initiate unknown events which lead to a neuropathy withdegeneration of long axons. These neuropathic organophosphates leave anegatively charged group covalently attached to the active-site serineresidue, which causes a toxic gain of function in NTE (Glynn, P. (1999)Biochem. J. 344:625-631). Further, the Drosophila neurodegeneration geneswiss-cheese encodes a neuronal protein involved in glia-neuroninteraction and is homologous to the above human NTE (Moser, M. et al.(2000) Mech. Dev. 90:279-282).

Chitinases are chitin-degrading enzymes present in a variety oforganisms and participate in processes including cell wall remodeling,defense and catabolism. Chitinase activity has been found in humanserum, leukocytes, granulocytes, and in association with fertilizedoocytes in mammals (Escott, G. M. (1995) Infect. Immunol. 63:4770-4773;DeSouza, M. M. (1995) Endocrinology 136:2485-2496). Glycolytic andproteolytic molecules in humans are associated with tissue damage inlung diseases and with increased tumorigenicity and metastatic potentialof cancers (Mulligan, M. S. (1993) Proc. Natl. Acad. Sci.90:11523-11527; Matrisian, L. M. (1991) Am. J. Med. Sci. 302:157-162;Witty, J. P. (1994) Cancer Res. 54:4805-4812). The discovery of a humanenzyme with chitinolytic activity is noteworthy given the lack ofendogenous chitin in the human body (Raghavan, N. (1994) Infect. Immun.62:1901-1908). However, there is a group of mammalian proteins thatshare homology with chitinases from various non-mammalian organisms,such as bacteria, fungi, plants, and insects. The members of this familydiffer in their ability to hydrolyze chitin or chitin-like substrates.Some of the mammalian members of the family, such as a bovine wheychitotriosidase and human cartilage proteins which do not demonstratespecific chitinolytic activity, are expressed in association with tissueremodeling events (Rejman, J. J. (1988) Biochem. Biophys. Res. Commun.150:329-334, Nyirkos, P. (1990) Biochem. J. 268:265-268). Elevatedlevels of human cartilage proteins have been reported in the synovialfluid and cartilage of patients with rheumatoid arthritis, a diseasewhich produces a severe degradation of the cartilage and a proliferationof the synovial membrane in the affected joints (Hakala, B. E. (1993) J.Biol. Chem. 268:25803-25810).

A small subclass of hydrolases acting on ether bonds includes thethioether hydrolases. S-adenosyl-L-homocysteine hydrolase, also known asAdoHcyase or SAHH (PROSITE PDOC00603; EC 3.3.1.1), is a thioetherhydrolase first described in rat liver extracts as the activityresponsible for the reversible hydrolysis of S-adenosyl-L-homocysteine(AdoHcy) to adenosine and homocysteine (Sganga, M. W. et al. (1992) PNAS89:6328-6332). SAHH is a cytosolic enzyme that has been found in allcells that have been tested, with the exception of Escherichia coli andcertain related bacteria (Walker, R. D. et al. (1975) Can. J. Biochem.53:312-319; Shimizu, S. et al. (1988) FEMS Microbiol. Lett. 51:177-180;Shimizu, S. et al. (1984) Eur. J. Biochem. 141:385-392). SAHH activityis dependent on NAD⁺ as a cofactor. Deficiency of SAHH is associatedwith hypermethioninemia (Online Mendelian Inheritance in Man (OMIM)#180960 Hypermethioninemia), a pathologic condition characterized byneonatal cholestasis, failure to thrive, mental and motor retardation,facial dysmorphism with abnormal hair and teeth, and myocaridopathy(Labrune, P. et al. (1990) J. Pediat. 117:220-226).

Another subclass of hydrolases includes those enzymes which act oncarbon-nitrogen (C—N) bonds other than peptide bonds. To this subclassbelong those enzymes hydrolyzing amides, amidines, and other C—N bonds.This subclass is further subdivided on the basis of substratespecificity such as linear amides, cyclic amides, linear amidines,cyclic amidines, nitrites and other compounds. A hydrolase belonging tothe sub-subclass of enzymes acting on the cyclic amidines is adenosinedeaminase (ADA). ADA catalyzes the breakdown of adenosine to inosine.ADA is present in many mammalian tissues, including placenta, muscle,lung, stomach, digestive diverticulum, spleen, erythrocytes, thymus,seminal plasma, thyroid, T-cells, bone marrow stem cells, and liver. Asubclass of ADAs, ADAR, act on RNA and are classified as RNA editases.An ADAR from Drosophila, DADAR, expressed in the developing nervoussystem, may act on para voltage-gated Na+ channel transcripts in thecentral nervous system (Palladino, M. J. et al. (2000) RNA 6:1004-1018).ADA deficiency causes profound lymphopenia with severe combinedimmunodeficiency (SCID). Cells from patients with ADA deficiency containlow, sometimes undetectable, amounts of ADA catalytic activity and ADAprotein. ADA deficiency stems from genetic mutations in the ADA gene(Hershfield, M. S. (1998) Semin. Hematol. 4:291-298). Metabolicconsequences of ADA deficiency are associated with defects inalveogenesis, pulmonary inflammation, and airway obstruction (Blackburn,M. R. et al. (2000) J. Exp. Med. 192:159-170).

Pancreatic ribonucleases (RNase) are pyrimidine-specific endonucleasesfound in high quantity in the pancreas of certain mammalian taxa and ofsome reptiles (Beintema, J. J. et al (1988) Prog. Biophys. Mol. Biol.51:165-192). Proteins in the mammalian pancreatic RNase superfamily arenoncytosolic endonucleases that degrade RNA through a two-steptransphosphorolytic-hydrolytic reaction (Beintema, J. J. et al. (1986)Mol. Biol. Evol. 3:262-275). Specifically, the enzymes are involved inendonucleolytic cleavage of 3′-phosphomononucleotides and3′-phosphooligonucleotides ending in C-P or U-P with 2′,3′-cyclicphosphate intermediates. Ribonucleases can unwind the DNA helix bycomplexing with single-stranded DNA; the complex arises by an extendedmulti-site cation-anion interaction between lysine and arginine residuesof the enzyme and phosphate groups of the nucleotides. Some of theenzymes belonging to this family appear to play a purely digestive role,whereas others exhibit potent and unusual biological activities(D'Alessio, G. (1993) Trends Cell Biol. 3:106-109). Proteins belongingto the pancreatic RNase family include: bovine seminal vesicle and brainribonucleases; kidney non-secretory ribonucleases (Beintema, J. J. et al(1986) FEBS Lett. 194:338-343); liver-type ribonucleases (Rosenberg, H.F. et al. (1989) PNAS U.S.A. 86:4460-4464); angiogenin, which inducesvascularisation of normal and malignant tissues; eosinophil cationicprotein (Hofsteenge, J. et al. (1989) Biochemistry 28:9806-9813), acytotoxin and helminthotoxin with ribonuclease activity; and frog liverribonuclease and frog sialic acid-binding lectin. The sequences ofpancreatic RNases contain 4 conserved disulfide bonds and 3 amino acidresidues involved in the catalytic activity.

ADP-ribosylation is a reversible post-translational protein modificationin which an ADP-ribose moiety is transferred from β-NAD to a targetamino acid such as arginine or cysteine. ADP-ribosylarginine hydrolasesregenerate arginine by removing ADP-ribose from the protein, completingthe ADP-ribosylation cycle (Moss, J. et al. (1997) Adv. Exp. Med. Biol.419:25-33). ADP-ribosylation is a well-known reaction among bacterialtoxins. Cholera toxin, for example, disrupts the adenylyl cyclase systemby ADP-ribosylating the α-subunit of the stimulatory G-protein, causingan increase in intracellular cAMP (Moss, J. and M. Vaughan (Eds) (1990)ADP-ribosylating Toxins and G-Proteins: Insights into SignalTransduction, American Society for Microbiology, Washington, D.C.).ADP-ribosylation may also have a regulatory function in eukaryotes,affecting such processes as cytoskeletal assembly (Zhou, H. et al.(1996) Arch. Biochem. Biophys. 334:214-222) and cell proliferation incytotoxic T-cells (Wang, J. et al. (1996) J. Immunol. 156:2819-2827).

Nucleotidases catalyze the formation of free nucleosides fromnucleotides. The cytosolic nucleotidase cN-I (5′ nucleotidase-I) clonedfrom pigeon heart catalyzes the formation of adenosine from AMPgenerated during ATP hydrolysis (Sala-Newby, G. B. et al. (1999) J.Biol. Chem. 274:17789-17793). Increased adenosine concentration isthought to be a signal of metabolic stress, and adenosine receptorsmediate effects including vasodilation, decreased stimulatory neuronfiring and ischemic preconditioning in the heart (Schrader, J. (1990)Circulation 81:389-391; Rubino, A. et al. (1992) Eur. J. Pharmacol.220:95-98; de Jong, J. W. et al. (2000) Pharmacol. Ther. 87:141-149).Deficiency of pyrimidine 5′-nucleotidase can result in hereditaryhemolytic anemia (OMIM #266120).

The lysozyme c superfamily consists of conventional lysozymes c,calcium-binding lysozymes c, and α-lactalbumin (Prager, E. M. and P.Jolles (1996) EXS 75:9-31). The proteins in this superfamily have 35-40%sequence homology and share a common three-dimensional fold, but canhave different functions. Lysozymes c are ubiquitous in a variety oftissues and secretions and can lyse the cell walls of certain bacteria(McKenzie, H. A. (1996) EXS 75:365-409). Alpha-lactalbumin is ametallo-protein that binds calcium and participates in the synthesis oflactose (Iyer, L. K. and P. K. Qasba (1999) Protein Eng. 12:129-139).Alpha-lactalbumin occurs in mammalian milk and colostrum (McKenzie,supra).

Lysozymes catalyze the hydrolysis of certain mucopolysaccharides ofbacterial cell walls, specifically, the beta (1-4) glycosidic linkagesbetween N-acetylmuramic acid and N-acetylglucosamine, and causebacterial lysis. Lysozymes occur in diverse organisms including viruses,birds, and mammals. In humans, lysozymes are found in spleen, lung,kidney, white blood cells, plasma, saliva, milk, tears, and cartilage(OMIM #153450 Lysozyme; Weaver, L. H. et al. (1985) J. Mol. Biol.184:739-741). Lysozyme c functions in ruminants as a digestive enzyme,releasing proteins from ingested bacterial cells, and may perform thesame function in human newborns (Braun, O. H. et al. (1995) Klin.Pediatr. 207:4-7).

The two known forms of lysozymes, chicken-type and goose-type, wereoriginally isolated from chicken and goose egg white, respectively.Chicken-type and goose-type lysozymes have similar three-dimensionalstructures, but different amino acid sequences (Nakano, T. and T. Graf(1991) Biochim. Biophys. Acta 1090:273-276). In chickens, both forms oflysozyme are found in neutrophil granulocytes (heterophils), but onlychicken-type lysozyme is found in egg white. Generally, chicken-typelysozyme mRNA is found in both adherent monocytes and macrophages andnonadherent promyelocytes and granulocytes as well as in cells of thebone marrow, spleen, bursa, and oviduct. Goose-type lysozyme mRNA isfound in non-adherent cells of the bone marrow and lung. Severalisozymes have been found in rabbits, including leukocytic,gastrointestinal, and possibly lymphoepithelial forms (OMIM #153450,supra; Nakano and Graf, supra; and GenBank GI 1310929). A human lysozymegene encoding a protein similar to chicken-type lysozyme has been cloned(Yoshimura, K. et al. (1988) Biochem. Biophys. Res. Commun.150:794-801). A consensus motif featuring regularly spaced cysteineresidues has been derived from the lysozyme C enzymes of various species(PROSITE PS00128). Lysozyme C shares about 40% amino acid sequenceidentity with α-lactalbumin.

Lysozymes have several disease associations. Lysozymuria is observed indiabetic nephropathy (Shima, M. et al. (1986) Clin. Chem. 32:1818-1822),endemic nephropathy (Bruckner, I. et al. (1978) Med. Interne.16:117-125), urinary tract infections (Heidegger, H. (1990) MinervaGinecol. 42:243-250), and acute monocytic leukemia (Shaw, M. T. (1978)Am. J. Hematol. 4:97-103). Nakano and Graf (supra) suggested a role forlysozyme in host defense systems. Older rabbits with an inheritedlysozyme deficiency show increased susceptibility to infections, such assubcutaneous abscesses (OMIM #153450, supra). Human lysozyme genemutations cause hereditary systemic amyloidosis, a rare autosomaldominant disease in which amyloid deposits form in the viscera,including the kidney, adrenal glands, spleen, and liver. This disease isusually fatal by the fifth decade. The amyloid deposits contain variantforms of lysozyme. Renal amyloidosis is the most common and potentiallythe most serious form of organ involvement (Pepys, M. B. et al. (1993)Nature 362:553-557; OMIM #105200 Familial Visceral Amyloidosis; Cotran,R. S. et al. (1994) Robbins Pathologic Basis of Disease, W.B. SaundersCompany, Philadelphia Pa., pp. 231-238). Increased levels of lysozymeand lactate have been observed in the cerebrospinal fluid of patientswith bacterial meningitis (Ponka, A. et al. (1983) Infection11:129-131). Acute monocytic leukemia is characterized by massivelysozymuria (Den Tandt, W. R. (1988) Int. J. Biochem. 20:713-719).

Lyases

Lyases are a class of enzymes that catalyze the cleavage of C—C, C—O,C—N, C—S, C-(halide), P—O, or other bonds without hydrolysis oroxidation to form two molecules, at least one of which contains a doublebond (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co., New YorkN.Y., p. 620). Under the International Classification of Enzymes (Webb,E. C. (1992) Enzyme Nomenclature 1992: Recommendations of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology on the Nomenclature and Classification of Enzymes,Academic Press, San Diego Calif.), lyases form a distinct classdesignated by the numeral 4 in the first digit of the enzyme number(i.e., EC 4.x.x.x).

Further classification of lyases reflects the type of bond cleaved aswell as the nature of the cleaved group. The group of C—C lyasesincludes carboxyl-lyases (decarboxylases), aldehyde-lyases (aldolases),oxo-acid-lyases, and other lyases. The C—O lyase group includeshydro-lyases, lyases acting on polysaccharides, and other lyases. TheC—N lyase group includes ammonia-lyases, amidine-lyases, amine-lyases(deaminases), and other lyases. Lyases are critical components ofcellular biochemistry, with roles in metabolic energy production,including fatty acid metabolism and the tricarboxylic acid cycle, aswell as other diverse enzymatic processes.

One important family of lyases are the carbonic anhydrases (CA), alsocalled carbonate dehydratases, which catalyze the hydration of carbondioxide in the reaction H₂O+CO₂≈HCO₃ ⁻+H⁺. CA accelerates this reactionby a factor of over 10⁶ by virtue of a zinc ion located in a deep cleftabout 15 Å below the protein's surface and co-ordinated to the imidazolegroups of three His residues. Water bound to the zinc ion is rapidlyconverted to HCO₃ ⁻.

Eight enzymatic and evolutionarily related forms of carbonic anhydraseare currently known to exist in humans: three cytosolic isozymes (CAI,CAII, and CAIII), two membrane-bound forms (CAIV and CAVII), amitochondrial form (CAV), a secreted salivary form (CAVI) and a yetuncharacterized isozyme (PROSITE PDOC00146 Eukaryotic-type carbonicanhydrases signature). Though the isoenzymes CAI, CAII, and bovine CAIIIhave similar secondary structures and polypeptide-chain folds, CAI has 6tryptophans, CAII has 7 and CAIII has 8 (Boren, K. et al. (1996) ProteinSci. 5:2479-2484). CAII is the predominant CA isoenzyme in the brain ofmammals.

CAs participate in a variety of physiological processes that involve pHregulation, CO₂ and HCO₃ ⁻ transport, ion transport, and water andelectrolyte balance. For example, CAII contributes to H⁺ secretion bygastric parietal cells, by renal tubular cells, and by osteoclasts thatsecrete H⁺ to acidify the bone-resorbing compartment. In addition, CAIIpromotes HCO₃ ⁻ secretion by pancreatic duct cells, cilary bodyepithelium, choroid plexus, salivary gland acinar cells, and distalcolonal epithelium, thus playing a role in the production of pancreaticjuice, aqueous humor, cerebrospinal fluid, and saliva, and contributingto electrolyte and water balance. CAII also promotes CO₂ exchange inproximal tubules in the kidney, in erythrocytes, and in lung. CAIV hasroles in several tissues: it facilitates HCO₃ ⁻ reabsorption in thekidney; promotes CO₂ flux in tissues including brain, skeletal muscle,and heart muscle; and promotes CO₂ exchange from the blood to thealveoli in the lung. CAVI probably plays a role in pH regulation insaliva, along with CAII, and may have a protective effect in theesophagus and stomach. Mitochondrial CAV appears to play important rolesin gluconeogenesis and ureagenesis, based on the effects of CAinhibitors on these pathways. (Sly, W. S. and P. Y. Hu (1995) Ann. Rev.Biochem. 64:375-401.)

A number of disease states are marked by variations in CA activity.Mutations in CAII which lead to CAII deficiency are the cause ofosteopetrosis with renal tubular acidosis (OMIM #259730 Osteopetrosiswith Renal Tubular Acidosis). The concentration of CAII in thecerebrospinal fluid (CSF) appears to mark disease activity in patientswith brain damage. High CA concentrations have been observed in patientswith brain infarction. Patients with transient ischemic attack, multiplesclerosis, or epilepsy usually have CAII concentrations in the normalrange, but higher CAII levels have been observed in the CSF of thosewith central nervous system infection, dementia, or trigeminal neuralgia(Parkkila, A. K. et al. (1997) Eur. J. Clin. Invest. 27:392-397).Colonic adenomas and adenocarcinomas have been observed to fail to stainfor CA, whereas non-neoplastic controls showed CAI and CAII in thecytoplasm of the columnar cells lining the upper half of colonic crypts.The neoplasms show staining patterns similar to less mature cells liningthe base of normal crypts (Gramlich T. L. et al. (1990) Arch. Pathol.Lab. Med. 114:415-419).

Therapeutic interventions in a number of diseases involve altering CAactivity. CA inhibitors such as acetazolamide are used in the treatmentof glaucoma (Stewart, W. C. (1999) Curr. Opin. Opthamol. 10:99-108),essential tremor and Parkinson's disease (Uitti, R. J. (1998) Geriatrics53:46-48, 53-57), intermittent ataxia (Singhvi, J. P. et al. (2000)Neurology India 48:78-80), and altitude related illnesses (Klocke, D. L.et al. (1998) Mayo Clin. Proc. 73:988-992).

CA activity can be particularly useful as an indicator of long-termdisease conditions, since the enzyme reacts relatively slowly tophysiological changes. CAI and zinc concentrations have been observed todecrease in hyperthyroid Graves' disease (Yoshida, K. (1996) Tohoku J.Exp. Med. 178:345-356) and glycosylated CAI is observed in diabetesmellitus (Kondo, T. et al. (1987) Clin. Chim. Acta 166:227-236). Apositive correlation has been observed between CAI and CAII reactivityand endometriosis (Brinton, D. A. et al. (1996) Ann. Clin. Lab. Sci.26:409-420; D'Cruz , O. J. et al. (1996) Fertil. Steril. 66:547-556).

Another important member of the lyase family is ornithine decarboxylase(ODC), the initial rate-limiting enzyme in polyamine biosynthesis. ODCcatalyses the transformation of ornithine into putrescine in thereaction L-ornithine≈putrescine+CO₂. Polyamines, which includeputrescine and the subsequent metabolic pathway products spermidine andspermine, are ubiquitous cell components essential for DNA synthesis,cell differentiation, and proliferation. Thus the polyamines play a keyrole in tumor proliferation (Medina, M. A. et al. (1999) Biochem.Pharmacol. 57:1341-1344).

ODC is a pyridoxal-5′-phosphate (PLP)-dependent enzyme which is activeas a homodimer. Conserved residues include those at the PLP binding siteand a stretch of glycine residues thought to be part of a substratebinding region (PROSITE PDOC00685 Orn/DAP/Arg decarboxylase family 2signatures). Mammalian ODCs also contain PEST regions, sequencefragments enriched in proline, glutamic acid, serine, and threonineresidues that act as signals for intracellular degradation (Nedina etal., supra).

Many chemical carcinogens and tumor promoters increase ODC levels andactivity. Several known oncogenes may increase ODC levels by enhancingtranscription of the ODC gene, and ODC itself may act as an oncogenewhen expressed at very high levels. A high level of ODC is found in anumber of precancerous conditions, and elevation of ODC levels has beenused as part of a screen for tumor-promoting compounds (Pegg, A. E. etal. (1995) J. Cell. Biochem. Suppl. 22:132-138).

Inhibitors of ODC have been used to treat tumors in animal models andhuman clinical trials, and have been shown to reduce development oftumors of the bladder, brain, esophagus, gastrointestinal tract, lung,oral cavity, mammary gland, stomach, skin and trachea (Pegg et al.,supra; McCann, P. P. and A. E. Pegg (1992) Pharmac. Ther. 54:195-215).ODC also shows promise as a target for chemoprevention (Pegg et al.,supra). ODC inhibitors have also been used to treat infections byAfrican trypanosomes, malaria, and Pneumocystis carinii, and arepotentially useful for treatment of autoimmune diseases such as lupusand rheumatoid arthritis (McCann and Pegg, supra).

Another family of pyridoxal-dependent decarboxylases are the group IIdecarboxylases. This family includes glutamate decarboxylase (GAD) whichcatalyzes the decarboxylation of glutamate into the neurotransmitterGABA; histidine decarboxylase (HDC), which catalyzes the decarboxylationof histidine to histamine; aromatic-L-amino-acid decarboxylase (DDC),also known as L-dopa decarboxylase or tryptophan decarboxylase, whichcatalyzes the decarboxylation of tryptophan to tryptamine and also actson 5-hydroxy-tryptophan and dihydroxyphenylalanine (L-dopa); andcysteine sulfinic acid decarboxylase (CSD), the rate-limiting enzyme inthe synthesis of taurine from cysteine (PROSITE PDOC00329DDC/GAD/HDC/TyrDC pyridoxal-phosphate attachment site). Taurine is anabundant sulfonic amino acid in brain and is thought to act as anosmoregulator in brain cells (Bitoun, M. and M. Tappaz (2000) J.Neurochem. 75:919-924).

Isomerases

Isomerases are a class of enzymes that catalyze geometric or structuralchanges within a molecule to form a single product. This class includesracemases and epimerases, cis-trans-isomerases, intramolecularoxidoreductases, intramolecular transferases (mutases) andintramolecular lyases. Isomerases are critical components of cellularbiochemistry with roles in metabolic energy production includingglycolysis, as well as other diverse enzymatic processes (Stryer, supra,pp. 483-507).

Racemases are a subset of isomerases that catalyze inversion of amolecule's configuration around the asymmetric carbon atom in asubstrate having a single center of asymmetry, thereby interconvertingtwo racemers. Epimerases are another subset of isomerases that catalyzeinversion of configuration around an asymmetric carbon atom in asubstrate with more than one center of symmetry, thereby interconvertingtwo epimers. Racemases and epimerases can act on amino acids andderivatives, hydroxy acids and derivatives, and carbohydrates andderivatives. The interconversion of UDP-galactose and UDP-glucose iscatalyzed by UDP-galactose-4′-epimerase. Proper regulation and functionof this epimerase is essential to the synthesis of glycoproteins andglycolipids. Elevated blood galactose levels have been correlated withUDP-galactose-4′-epimerase deficiency in screening programs of infants(Gitzelmann, R. (1972) Helv. Paediat. Acta 27:125-130).

Correct folding of newly synthesized proteins is assisted by molecularchaperones and folding catalysts, two unrelated groups of helpermolecules. Chaperones suppress non-productive side reactions bystoichiometric binding to folding intermediates, whereas folding enzymescatalyze some of the multiple folding steps that enable proteins toattain their final functional configurations (Kern, G. et al. (1994)FEBS Lett. 348:145-148). One class of folding enzymes, the peptidylprolyl cis-trans isomerases (PPIases), isomerizes certain proline imidicbonds in what is considered to be a rate limiting step in proteinmaturation and export. PPIases catalyze the cis to trans isomerizationof certain proline imidic bonds in proteins. There are threeevolutionarily unrelated families of PPIases: the cyclophilins, theFK506 binding proteins, and the newly characterized parvulin family(Rahfeld, J. U. et al. (1994) FEBS Lett. 352:180-184).

The cyclophilins (CyP) were originally identified as major receptors forthe immunosuppressive drug cyclosporin A (CsA), an inhibitor of T-cellactivation (Handschumacher, R. E. et al. (1984) Science 226:544-547;Harding, M. W. et al. (1986) J. Biol. Chem. 261:8547-8555). Thus, thepeptidyl-prolyl isomerase activity of CyP may be part of the signalingpathway that leads to T-cell activation. Subsequent work demonstratedthat CyP's isomerase activity is essential for correct protein foldingand/or protein trafficking, and may also be involved inassembly/disassembly of protein complexes and regulation of proteinactivity. For example, in Drosophila, the CyP NinaA is required forcorrect localization of rhodopsins, while a mammalian CyP (Cyp40) ispart of the Hsp90/Hsp70 complex that binds steroid receptors. Themammalian CyP (CypA) has been shown to bind the gag protein from humanimmunodeficiency virus 1 (HIV-1), an interaction that can be inhibitedby cyclosporin. Since cyclosporin has potent anti-HIV-1 activity, CypAmay play an essential function in HIV-1 replication. Finally, Cyp40 hasbeen shown to bind and inactivate the transcription factor c-Myb, aneffect that is reversed by cyclosporin. This effect implicates CyP inthe regulation of transcription, transformation, and differentiation(Bergsma, D. J. et al (1991) J. Biol. Chem. 266:23204-23214; Hunter, T.(1998) Cell 92:141-143; and Leverson, J. D. and S. A. Ness (1998) Mol.Cell. 1:203-211).

One of the major rate limiting steps in protein folding is thethiol:disulfide exchange that is necessary for correct protein assembly.Although incubation of reduced, unfolded proteins in buffers withdefined ratios of oxidized and reduced thiols can lead to nativeconformation, the rate of folding is slow and the attainment of nativeconformation decreases proportionately with the size and number ofcysteines in the protein. Certain cellular compartments such as theendoplasmic reticulum of eukaryotes and the periplasmic space ofprokaryotes are maintained in a more oxidized state than the surroundingcytosol. Correct disulfide formation can occur in these compartments,but at a rate that is insufficient for normal cell processes andinadequate for synthesizing secreted proteins. The protein disulfideisomerases, thioredoxins and glutaredoxins are able to catalyze theformation of disulfide bonds and regulate the redox environment in cellsto enable the necessary thiol:disulfide exchanges (Loferer, H. (1995) J.Biol. Chem. 270:26178-26183).

Each of these proteins has somewhat different functions, but all belongto a group of disulfide-containing redox proteins that contain aconserved active-site sequence and are ubiquitously distributed ineukaryotes and prokaryotes. Protein disulfide isomerases are found inthe endoplasmic reticulum of eukaryotes and in the periplasmic space ofprokaryotes. They function by exchanging their own disulfide for a thiolin a folding peptide chain. In contrast, the reduced thioredoxins andglutaredoxins are generally found in the cytoplasm and function bydirectly reducing disulfides in the substrate proteins.

Oxidoreductases can be isomerases as well. Oxidoreductases catalyze thereversible transfer of electrons from a substrate that becomes oxidizedto a substrate that becomes reduced. This class of enzymes includesdehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, andreductases. Proper maintenance of oxidoreductase levels isphysiologically important. For example, genetically-linked deficienciesin lipoamide dehydrogenase can result in lactic acidosis (Robinson, B.H. et al. (1977) Pediat. Res. 11:1198-1202).

Another subgroup of isomerases are the transferases (or mutases).Transferases transfer a chemical group from one compound (the donor) toanother compound (the acceptor). The types of groups transferred bythese enzymes include acyl groups, amino groups, phosphate groups(phosphotransferases or phosphomutases), and others. The transferasecarnitine palmitoyltransferase is an important component of fatty acidmetabolism. Genetically-linked deficiencies in this transferase can leadto myopathy (Scriver, C. et al. (1995) The Metabolic and Molecular Basisof Inherited Disease, McGraw-Hill, New York N.Y., pp. 1501-1533).

Yet another subgroup of isomerases are the topoisomersases.Topoisomerases are enzymes that affect the topological state of DNA. Forexample, defects in topoisomerases or their regulation can affect normalphysiology. Reduced levels of topoisomerase II have been correlated withsome of the DNA processing defects associated with the disorderataxia-telangiectasia (Singh, S. P. et al. (1988) Nucleic Acids Res.16:3919-3929).

Ligases

Ligases catalyze the formation of a bond between two substratemolecules. The process involves the hydrolysis of a pyrophosphate bondin ATP or a similar energy donor. Ligases are classified based on thenature of the type of bond they form, which can include carbon-oxygen,carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric esterbonds.

Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA(tRNA) synthetases which are important RNA-associated enzymes with rolesin translation. Protein biosynthesis depends on each amino acid forminga linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases areresponsible for the activation and correct attachment of an amino acidwith its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can bedivided into two structural classes, and each class is characterized bya distinctive topology of the catalytic domain. Class I enzymes containa catalytic domain based on the nucleotide-binding “Rossman fold”. ClassII enzymes contain a central catalytic domain, which consists of aseven-stranded antiparallel β-sheet motif, as well as N- and C-terminalregulatory domains. Class II enzymes are separated into two groups basedon the heterodimeric or homodimeric structure of the enzyme; the lattergroup is further subdivided by the structure of the N- and C-terminalregulatory domains (Hartlein, M. and S. Cusack, (1995) J. Mol. Evol.40:519-530). Autoantibodies against aminoacyl-tRNAs are generated bypatients with dermatomyositis and polymyositis, and correlate stronglywith complicating interstitial lung disease (ILD). These antibodiesappear to be generated in response to viral infection, and coxsackievirus has been used to induce experimental viral myositis in animals.

Ligases forming carbon-sulfur bonds (acid-thiol ligases) mediate a largenumber of cellular biosynthetic intermediary metabolism processesinvolving intermolecular transfer of carbon atom-containing substrates(carbon substrates). Examples of such reactions include thetricarboxylic acid cycle, synthesis of fatty acids and long-chainphospholipids, synthesis of alcohols and aldehydes, synthesis ofintermediary metabolites, and reactions involved in the amino aciddegradation pathways. Some of these reactions require input of energy,usually in the form of conversion of ATP to either ADP or AMP andpyrophosphate.

In many cases, a carbon substrate is derived from a small moleculecontaining at least two carbon atoms. The carbon substrate is oftencovalently bound to a larger molecule which acts as a carbon substratecarrier molecule within the cell. In the biosynthetic mechanismsdescribed above, the carrier molecule is coenzyme A. Coenzyme A (CoA) isstructurally related to derivatives of the nucleotide ADP and consistsof 4′-phosphopantetheine linked via a phosphodiester bond to the alphaphosphate group of adenosine 3′,5′-bisphosphate. The terminal thiolgroup of 4′-phosphopantetheine acts as the site for carbon substratebond formation. The predominant carbon substrates which utilize CoA as acarrier molecule during biosynthesis and intermediary metabolism in thecell are acetyl, succinyl, and propionyl moieties, collectively referredto as acyl groups. Other carbon substrates include enoyl lipid, whichacts as a fatty acid oxidation intermediate, and carnitine, which actsas an acetyl-CoA flux regulator/mitochondrial acyl group transferprotein. Acyl-CoA and acetyl-CoA are synthesized in the cell by acyl-CoAsynthetase and acetyl-CoA synthetase, respectively.

Activation of fatty acids is mediated by at least three forms ofacyl-CoA synthetase activity: i) acetyl-CoA synthetase, which activatesacetate and several other low molecular weight carboxylic acids and isfound in muscle mitochondria and the cytosol of other tissues; ii)medium-chain acyl-CoA synthetase, which activates fatty acids containingbetween four and eleven carbon atoms (predominantly from dietarysources), and is present only in liver mitochondria; and iii) acyl CoAsynthetase, which is specific for long chain fatty acids with betweensix and twenty carbon atoms, and is found in microsomes and themitochondria. Proteins associated with acyl-CoA synthetase activity havebeen identified from many sources including bacteria, yeast, plants,mouse, and man. The activity of acyl-CoA synthetase may be modulated byphosphorylation of the enzyme by cAMP-dependent protein kinase.

Ligases forming carbon-nitrogen bonds include amide synthases such asglutamine synthetase (glutamate-ammonia ligase) that catalyzes theamination of glutamic acid to glutamine by ammonia using the energy ofATP hydrolysis. Glutamine is the primary source for the amino group invarious amide transfer reactions involved in de novo pyrimidinenucleotide synthesis and in purine and pyrimidine ribonucleotideinterconversions. Overexpression of glutamine synthetase has beenobserved in primary liver cancer (Christa, L. et al. (1994) Gastroent.106:1312-1320).

Acid-amino-acid ligases (peptide synthases) are represented by theubiquitin conjugating enzymes which are associated with the ubiquitinconjugation system (UCS), a major pathway for the degradation ofcellular proteins in eukaryotic cells and some bacteria. The UCSmediates the elimination of abnormal proteins and regulates thehalf-lives of important regulatory proteins that control cellularprocesses such as gene transcription and cell cycle progression. In theUCS pathway, proteins targeted for degradation are conjugated toubiquitin (Ub), a small heat stable protein. Ub is first activated by aubiquitin-activating enzyme (E1), and then transferred to one of severalUb-conjugating enzymes (E2). E2 then links the Ub molecule through itsC-terminal glycine to an internal lysine (acceptor lysine) of a targetprotein. The ubiquitinated protein is then recognized and degraded byproteasome, a large, multisubunit proteolytic enzyme complex, andubiquitin is released for reutilization by ubiquitin protease. The UCSis implicated in the degradation of mitotic cyclic kinases,oncoproteins, tumor suppressor genes such as p53, viral proteins, cellsurface receptors associated with signal transduction, transcriptionalregulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell79:13-21).

Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymesand enzyme complexes that participate in the de novo pathways of purineand pyrimidine biosynthesis. Because these pathways are critical to thesynthesis of nucleotides for replication of both RNA and DNA, many ofthese enzymes have been the targets of clinical agents for the treatmentof cell proliferative disorders such as cancer and infectious diseases.

Purine biosynthesis occurs de novo from the amino acids glycine andglutamine, and other small molecules. Three of the key reactions in thisprocess are catalyzed by a trifunctional enzyme composed ofglycinamide-ribonucleotide synthetase (GARS), aminoimidazoleribonucleotide synthetase (AIRS), and glycinamide ribonucleotidetransformylase (GART). Together these three enzymes combine ribosylaminephosphate with glycine to yield phosphoribosyl aminoimidazole, aprecursor to both adenylate and guanylate nucleotides. Thistrifunctional protein has been implicated in the pathology of Downssyndrome (Aimi, J. et al. (1990) Nucleic Acid Res. 18:6665-6672).Adenylosuccinate synthetase catalyzes a later step in purinebiosynthesis that converts inosinic acid to adenylosuccinate, a key stepon the path to ATP synthesis. This enzyme is also similar to anothercarbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes asimilar reaction in the urea cycle (Powell, S. M. et al. (1992) FEBSLett. 303:4-10).

Adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminasemay be considered as a functional unit, the purine nucleotide cycle.This cycle converts AMP to inosine monophosphate (IMP) and reconvertsIMP to AMP via adenylosuccinate, thereby producing NH₃ and formingfumarate from aspartate. In muscle, the purine nucleotide cyclefunctions, during intense exercise, in the regeneration of ATP bypulling the adenylate kinase reaction in the direction of ATP formationand by providing Krebs cycle intermediates. In kidney, the purinenucleotide cycle accounts for the release of NH₃ under normal acid-baseconditions. In brain, the purine nucleotide cycle may contribute to ATPrecovery. Adenylosuccinate lyase deficiency provokes psychomotorretardation, often accompanied by autistic features (Van den Berghe, G.et al. (1992) Prog Neurobiol. 39:547-561). A marked imbalance in theenzymic pattern of purine metabolism is linked with transformationand/or progression in cancer cells. In rat hepatomas the specificactivities of the anabolic enzymes, IMP dehydrogenase, GMP synthetase,adenylosuccinate synthetase, adenylosuccinase, AMP deaminase andamidophosphoribosyltransferase, increased to 13.5-, 3.7-, 3.1-, 1.8-,5.5- and 2.8-fold, respectively, of those in normal liver (Weber, G.(1983) Clin. Biochem. 16:57-63).

Like the de novo biosynthesis of purines, de novo synthesis of thepyrimidine nucleotides uridylate and cytidylate also arises from acommon precursor, in this instance the nucleotide orotidylate derivedfrom orotate and phosphoribosyl pyrophosphate (PPRP). Again atrifunctional enzyme comprising three carbon-nitrogen ligases plays akey role in the process. In this case the enzymes aspartatetranscarbamylase (ATCase), carbamyl phosphate synthetase II, anddihydroorotase (DHOase) are encoded by a single gene called CAD.Together these three enzymes combine the initial reactants in pyrimidinebiosynthesis, glutamine, CO₂ and ATP to form dihydroorotate, theprecursor to orotate and orotidylate (Iwahana, H. et al. (1996) Biochem.Biophys. Res. Commun. 219:249-255). Further steps then lead to thesynthesis of uridine nucleotides from orotidylate. Cytidine nucleotidesare derived from uridine-5′-triphosphate (UTP) by the amidation of UTPusing glutamine as the amino donor and the enzyme CTP synthetase.Regulatory mutations in the human CTP synthetase are believed to confermulti-drug resistance to agents widely used in cancer therapy (Yamauchi,M. et al. (1990) EMBO J. 9:2095-2099).

Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoAcarboxylase and pyruvate carboxylase. Acetyl-CoA carboxylase catalyzesthe carboxylation of acetyl-CoA from CO₂ and H₂O using the energy of ATPhydrolysis. Acetyl-CoA carboxylase is the rate-limiting enzyme in thebiogenesis of long-chain fatty acids. Two isoforms of acetyl-CoAcarboxylase, types I and types II, are expressed in human in atissue-specific manner (Ha, J. et al. (1994) Eur. J. Biochem.219:297-306). Pyruvate carboxylase is a nuclear-encoded mitochondrialenzyme that catalyzes the conversion of pyruvate to oxaloacetate, a keyintermediate in the citric acid cycle.

Ligases forming phosphoric ester bonds include the DNA ligases involvedin both DNA replication and repair. DNA ligases seal phosphodiesterbonds between two adjacent nucleotides in a DNA chain using the energyfrom ATP hydrolysis to first activate the free 5′-phosphate of onenucleotide and then react it with the 3′-OH group of the adjacentnucleotide. This resealing reaction is used in DNA replication to joinsmall DNA fragments called “Okazaki” fragments that are transientlyformed in the process of replicating new DNA, and in DNA repair. DNArepair is the process by which accidental base changes, such as thoseproduced by oxidative damage, hydrolytic attack, or uncontrolledmethylation of DNA, are corrected before replication or transcription ofthe DNA can occur. Bloom's syndrome is an inherited human disease inwhich individuals are partially deficient in DNA ligation andconsequently have an increased incidence of cancer (Alberts et al.,supra, p. 247).

Pantothenate synthetase (D-pantoate; beta-alanine ligase (AMP-forming);EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitaminB(5)) synthesis. It catalyzes the condensation of pantoate withbeta-alanine in an ATP-dependent reaction. The enzyme is dimeric, withtwo well-defined domains per protomer: the N-terminal domain, a Rossmannfold, contains the active site cavity, with the C-terminal domainforming a hinged lid. The N-terminal domain is structurally very similarto class I aminoacyl-tRNA synthetases and is thus a member of thecytidylyltransferase superfamily (von Delft, F. et al. (2000) Structure(Camb) 9:439-450).

Farnesyl diphosphate synthase (FPPS) is an essential enzyme that isrequired both for cholesterol synthesis and protein prenylation. Theenzyme catalyzes the formation of farnesyl diphosphate fromdimethylallyl diphosphate and isopentyl diphosphate. FPPS is inhibitedby nitrogen-containing biphosphonates, which can lead to the inhibitionof osteoclast-mediated bone resorption by preventing protein prenylation(Dunford, J. E. et al. (2001) J. Pharmacol. Exp. Ther. 296:235-242).

5-aminolevulinate synthase (ALAS; delta-aminolevulinate synthase; EC2.3.1.37) catalyzes the rate-limiting step in heme biosynthesis in botherythroid and non-erythroid tissues. This enzyme is unique in the hemebiosynthetic pathway in being encoded by two genes, the first encodingALAS1, the non-erythroid specific enzyme which is ubiquitouslyexpressed, and the second encoding ALAS2, which is expressed exclusivelyin erythroid cells. The genes for ALAS1 and ALAS2 are located,respectively, on chromosome 3 and on the X chromosome. Defects in thegene encoding ALAS2 result in X-linked sideroblastic anemia. Elevatedlevels of ALAS are seen in acute hepatic porphyrias and can be loweredby zinc mesoporphyrin.

Drug Metabolizing Enzymes (DMEs)

The metabolism of a drug and its movement through the body(pharmacokinetics) are important in determining its effects, toxicity,and interactions with other drugs. The three processes governingpharmacokinetics are the absorption of the drug, distribution to varioustissues, and elimination of drug metabolites. These processes areintimately coupled to drug metabolism, since a variety of metabolicmodifications alter most of the physicochemical and pharmacologicalproperties of drugs, including solubility, binding to receptors, andexcretion rates. The metabolic pathways which modify drugs also accept avariety of naturally occurring substrates such as steroids, fatty acids,prostaglandins, leukotrienes, and vitamins. The enzymes in thesepathways are therefore important sites of biochemical andpharmacological interaction between natural compounds, drugs,carcinogens, mutagens, and xenobiotics. It has long been appreciatedthat inherited differences in drug metabolism lead to drasticallydifferent levels of drug efficacy and toxicity among individuals.Advances in pharmacogenomics research, of which DMEs constitute animportant part, are promising to expand the tools and information thatcan be brought to bear on questions of drug efficacy and toxicity (SeeEvans, W. E. and R. V. Relling (1999) Science 286:487-491). DMEs havebroad substrate specificities, unlike antibodies, for example, which arediverse and highly specific. Since DMEs metabolize a wide variety ofmolecules, drug interactions may occur at the level of metabolism sothat, for example, one compound may induce a DME that affects themetabolism of another compound.

Drug metabolic reactions are categorized as Phase I, which prepare thedrug molecule for functioning and further metabolism, and Phase II,which are conjugative. In general, Phase I reaction products arepartially or fully inactive, and Phase II reaction products are thechief excreted species. However, Phase I reaction products are sometimesmore active than the original administered drugs; this metabolicactivation principle is exploited by pro-drugs (e.g. L-dopa).Additionally, some nontoxic compounds (e.g. aflatoxin, benzo[α]pyrene)are metabolized to toxic intermediates through these pathways. Phase Ireactions are usually rate-limiting in drug metabolism. Prior exposureto the compound, or other compounds, can induce the expression of PhaseI enzymes however, and thereby increase substrate flux through themetabolic pathways. (See Klaassen, C. D. et al. (1996) Casarett andDoull's Toxicology: The Basic Science of Poisons, McGraw-Hill, New York,N.Y., pp. 113-186; Katzung, B. G. (1995) Basic and ClinicalPharmacology, Appleton and Lange, Norwalk, Conn., pp. 48-59; Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabolism, Blackie Academicand Professional, London.).

The major classes of Phase I enzymes include, but are not limited to,cytochrome P450 and flavin-containing monooxygenase. Other enzymeclasses involved in Phase I-type catalytic cycles and reactions include,but are not limited to, NADPH cytochrome P450 reductase (CPR), themicrosomal cytochrome b5/NADH cytochrome b5 reductase system, theferredoxin/ferredoxin reductase redox pair, aldo/keto reductases, andalcohol dehydrogenases. The major classes of Phase II enzymes include,but are not limited to, UDP glucuronyltransferase, sulfotransferase,glutathione S-transferase, N-acyltransferase, and N-acetyl transferase.

Cytochrome P450 and P450 Catalytic Cycle-Associated Enzymes

Members of the cytochrome P450 superfamily of enzymes catalyze theoxidative metabolism of a variety of substrates, including naturalcompounds such as steroids, fatty acids, prostaglandins, leukotrienes,and vitamins, as well as drugs, carcinogens, mutagens, and xenobiotics.Cytochromes P450, also known as P450 heme-thiolate proteins, usually actas terminal oxidases in multi-component electron transfer chains, calledP450-containing monooxygenase systems. Specific reactions catalyzedinclude hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-,and O-dealkylations, desulfation, deamination, and reduction of azo,nitro, and N-oxide groups. These reactions are involved insteroidogenesis of glucocorticoids, cortisols, estrogens, and androgensin animals; insecticide resistance in insects; herbicide resistance andflower coloring in plants; and environmental bioremediation bymicroorganisms. Cytochrome P450 actions on drugs, carcinogens, mutagens,and xenobiotics can result in detoxification or in conversion of thesubstance to a more toxic product. Cytochromes P450 are abundant in theliver, but also occur in other tissues; the enzymes are located inmicrosomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite PDOC00081Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450IE-Class P450 Group I signature; Graham-Lorence, S. and J. A. Peterson(1996) FASEB J. 10:206-214.)

Four hundred cytochromes P450 have been identified in diverse organismsincluding bacteria, fungi, plants, and animals (Graham-Lorence andPeterson, supra). The B-class is found in prokaryotes and fungi, whilethe E-class is found in bacteria, plants, insects, vertebrates, andmammals. Five subclasses or groups are found within the larger family ofE-class cytochromes P450 (PRINTS EP450I E-Class P450 Group I signature).

All cytochromes P450 use a heme cofactor and share structuralattributes. Most cytochromes P450 are 400 to 530 amino acids in length.The secondary structure of the enzyme is about 70% alpha-helical andabout 22% beta-sheet. The region around the heme-binding site in theC-terminal part of the protein is conserved among cytochromes P450. Aten amino acid signature sequence in this heme-iron ligand region hasbeen identified which includes a conserved cysteine involved in bindingthe heme iron in the fifth coordination site. In eukaryotic cytochromesP450, a membrane-spanning region is usually found in the first 15-20amino acids of the protein, generally consisting of approximately 15hydrophobic residues followed by a positively charged residue. (SeeProsite PDOC00081, supra; Graham-Lorence and Peterson, supra.)

Cytochrome P450 enzymes are involved in cell proliferation anddevelopment. The enzymes have roles in chemical mutagenesis andcarcinogenesis by metabolizing chemicals to reactive intermediates thatform adducts with DNA (Nebert, D. W. and F. J. Gonzalez (1987) Ann. Rev.Biochem. 56:945-993). These adducts can cause nucleotide changes and DNArearrangements that lead to oncogenesis. Cytochrome P450 expression inliver and other tissues is induced by xenobiotics such as polycyclicaromatic hydrocarbons, peroxisomal proliferators, phenobarbital, and theglucocorticoid dexamethasone (Dogra, S. C. et al. (1998) Clin. Exp.Pharmacol. Physiol. 25:1-9). A cytochrome P450 protein may participatein eye development as mutations in the P450 gene CYP1B1 cause primarycongenital glaucoma (OMIM #601771 Cytochrome P450, subfamily I(dioxin-inducible), polypeptide 1; CYP1B1).

Cytochromes P450 are associated with inflammation and infection. Hepaticcytochrome P450 activities are profoundly affected by various infectionsand inflammatory stimuli, some of which are suppressed and some induced(Morgan, E. T. (1997) Drug Metab. Rev. 29:1129-1188). Effects observedin vivo can be mimicked by proinflammatory cytokines and interferons.Autoantibodies to two cytochrome P450 proteins were found in patientswith autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy(APECED), a polyglandular autoimmune syndrome (OMIM #240300 Autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy).

Mutations in cytochromes P450 have been linked to metabolic disorders,including congenital adrenal hyperplasia, the most common adrenaldisorder of infancy and childhood; pseudovitamin D-deficiency rickets;cerebrotendinous xanthomatosis, a lipid storage disease characterized byprogressive neurologic dysfunction, premature atherosclerosis, andcataracts; and an inherited resistance to the anticoagulant drugscoumarin and warfarin (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, Inc. New York, N.Y., pp.1968-1970; Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka,S. et al. (1998) N. Engl. J. Med. 338:653-661; OMIM #213700Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).Extremely high levels of expression of the cytochrome P450 proteinaromatase were found in a fibrolamellar hepatocellular carcinoma from aboy with severe gynecomastia (feminization) (Agarwal, V. R. (1998) J.Clin. Endocrinol. Metab. 83:1797-1800).

The cytochrome P450 catalytic cycle is completed through reduction ofcytochrome P450 by NADPH cytochrome P450 reductase (CPR). Anothermicrosomal electron transport system consisting of cytochrome b5 andNADPH cytochrome b5 reductase has been widely viewed as a minorcontributor of electrons to the cytochrome P450 catalytic cycle.However, a recent report by Lamb, D. C. et al. (1999; FEBS Lett.462:283-288) identifies a Candida albicans cytochrome P450 (CYP51) whichcan be efficiently reduced and supported by the microsomal cytochromeb5/NADPH cytochrome b5 reductase system. Therefore, there are likelymany cytochromes P450 which are supported by this alternative electrondonor system.

Cytochrome b5 reductase is also responsible for the reduction ofoxidized hemoglobin (methemoglobin, or ferrihemoglobin, which is unableto carry oxygen) to the active hemoglobin (ferrohemoglobin) in red bloodcells. Methemoglobinemia results when there is a high level of oxidantdrugs or an abnormal hemoglobin (hemoglobin M) which is not efficientlyreduced. Methemoglobinemia can also result from a hereditary deficiencyin red cell cytochrome b5 reductase (Reviewed in Mansour, A. and A. A.Lurie (1993) Am. J. Hematol. 42:7-12).

Members of the cytochrome P450 family are also closely associated withvitamin D synthesis and catabolism. Vitamin D exists as two biologicallyequivalent prohormones, ergocalciferol (vitamin D₂), produced in planttissues, and cholecalciferol (vitamin D₃), produced in animal tissues.The latter form, cholecalciferol, is formed upon the exposure of7-dehydrocholesterol to near ultraviolet light (i.e., 290-310 nm),normally resulting from even minimal periods of skin exposure tosunlight (reviewed in Miller, W. L. and A. A. Portale (2000) TrendsEndocrinol. Metab. 11:315-319).

Both prohormone forms are further metabolized in the liver to25-hydroxyvitamin D (25(OH)D) by the enzyme 25-hydroxylase. 25(OH)D isthe most abundant precursor form of vitamin D which must be furthermetabolized in the kidney to the active form, 1α,25-dihydroxyvitamin D(1α,25(OH)₂D), by the enzyme 25-hydroxyvitamin D 1α-hydroxylase(1α-hydroxylase). Regulation of 1α,25(OH)₂D production is primarily atthis final step in the synthetic pathway. The activity of 1α-hydroxylasedepends upon several physiological factors including the circulatinglevel of the enzyme product (1α,25(OH)₂D) and the levels of parathyroidhormone (PTH), calcitonin, insulin, calcium, phosphorus, growth hormone,and prolactin. Furthermore, extrarenal 1α-hydroxylase activity has beenreported, suggesting that tissue-specific, local regulation of1α,25(OH)2D production may also be biologically important. The catalysisof 1α,25(OH)₂D to 24,25-dihydroxyvitamin D (24,25(OH)₂D), involving theenzyme 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase), also occursin the kidney. 24-hydroxylase can also use 25(OH)D as a substrate(Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925;Miller and Portale, supra; and references within).

Vitamin D 25-hydroxylase, 1α-hydroxylase, and 24-hydroxylase are allNADPH-dependent, type I (mitochondrial) cytochrome P450 enzymes thatshow a high degree of homology with other members of the family. VitaminD 25-hydroxylase also shows a broad substrate specificity and may alsoperform 26-hydroxylation of bile acid intermediates and 25, 26, and27-hydroxylation of cholesterol (Dilworth, F. J. et al. (1995) J. Biol.Chem. 270:16766-16774; Miller and Portale, supra; and referenceswithin).

The active form of vitamin D (1α,25(OH)₂D) is involved in calcium andphosphate homeostasis and promotes the differentiation of myeloid andskin cells. Vitamin D deficiency resulting from deficiencies in theenzymes involved in vitamin D metabolism (e.g., 1α-hydroxylase) causeshypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive)rickets, a disease characterized by loss of bone density and distinctiveclinical features, including bandy or bow leggedness accompanied by awaddling gait. Deficiencies in vitamin D 25-hydroxylase causecerebrotendinous xanthomatosis, a lipid-storage disease characterized bythe deposition of cholesterol and cholestanol in the Achilles' tendons,brain, lungs, and many other tissues. The disease presents withprogressive neurologic dysfunction, including postpubescent cerebellarataxia, atherosclerosis, and cataracts. Vitamin D 25-hydroxylasedeficiency does not result in rickets, suggesting the existence ofalternative pathways for the synthesis of 25(OH)D (Griffin, J. E. and J.B. Zerwekh (1983) J. Clin. Invest. 72:1190-1199; Gamblin, G. T. et al.(1985) J. Clin. Invest. 75:954-960; and Miller and Portale, supra).

Ferredoxin and ferredoxin reductase are electron transport accessoryproteins which support at least one human cytochrome P450 species,cytochrome P450c27 encoded by the CYP27 gene (Dilworth, F. J. et al.(1996) Biochem. J. 320:267-71). A Streptomyces griseus cytochrome P450,CYP104D1, was heterologously expressed in Escherichia coli and found tobe reduced by the endogenous ferredoxin and ferredoxin reductase enzymes(Taylor, M. et al. (1999) Biochem. Biophys. Res. Commun. 263:838-842),suggesting that many cytochrome P450 species may be supported by theferredoxin/ferredoxin reductase pair. Ferredoxin reductase has also beenfound in a model drug metabolism system to reduce actinomycin D, anantitumor antibiotic, to a reactive free radical species (Flitter, W. D.and R. P. Mason (1988) Arch. Biochem. Biophys. 267:632-639).

Flavin-Containing Monooxygnase (FMO)

Flavin-containing monooxygenases oxidize the nucleophilic nitrogen,sulfur, and phosphorus heteroatom of an exceptional range of substrates.Like cytochromes P450, FMOs are microsomal and use NADPH and O₂; thereis also a great deal of substrate overlap with cytochromes P450. Thetissue distribution of FMOs includes liver, kidney, and lung.

Isoforms of FMO in mammals include FMO1, FMO2, FMO3, FMO4, and FMO5,which are expressed in a tissue-specific manner. The isoforms differ intheir substrate specificities and properties such as inhibition byvarious compounds and stereospecificity of reaction. FMOs have a 13amino acid signature sequence, the components of which span theN-terminal two-thirds of the sequences and include the FAD bindingregion and the FATGY motif found in many N-hydroxylating enzymes (Stehr,M. et al. (1998) Trends Biochem. Sci. 23:56-57; PRINTS FMOXYGENASEFlavin-containing monooxygenase signature). Specific reactions includeoxidation of nucleophilic tertiary amines to N-oxides, secondary aminesto hydroxylamines and nitrones, primary amines to hydroxylamines andoximes, and sulfur-containing compounds and phosphines to S- andP-oxides. Hydrazines, iodides, selenides, and boron-containing compoundsare also substrates. FMOs are more heat labile and lessdetergent-sensitive than cytochromes P450 in vitro though FMO isoformsvary in thermal stability and detergent sensitivity.

FMOs play important roles in the metabolism of several drugs andxenobiotics. FMO (FMO3 in liver) is predominantly responsible formetabolizing (S)-nicotine to (S)-nicotine N-1′-oxide, which is excretedin urine. FMO is also involved in S-oxygenation of cimetidine, anH₂-antagonist widely used for the treatment of gastric ulcers.Liver-expressed forms of FMO are not under the same regulatory controlas cytochrome P450. In rats, for example, phenobarbital treatment leadsto the induction of cytochrome P450, but the repression of FMO1.

Lysyl Oxidase

Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidaseinvolved in the formation of connective tissue matrices by crosslinkingcollagen and elastin. LO is secreted as an N-glycosylated precursorprotein of approximately 50 kDa and cleaved to the mature form of theenzyme by a metalloprotease, although the precursor form is also active.The copper atom in LO is involved in the transport of electrons to andfrom oxygen to facilitate the oxidative deamination of lysine residuesin these extracellular matrix proteins. While the coordination of copperis essential to LO activity, insufficient dietary intake of copper doesnot influence the expression of the apoenzyme. However, the absence ofthe functional LO is linked to the skeletal and vascular tissuedisorders that are associated with dietary copper deficiency. LO is alsoinhibited by a variety of semicarbazides, hydrazines, and aminonitrites, as well as heparin. Beta-aminopropionitrile is a commonly usedinhibitor. LO activity is increased in response to ozone, cadmium, andelevated levels of hormones released in response to local tissue trauma,such as transforming growth factor-beta, platelet-derived growth factor,angiotensin II, and fibroblast growth factor. Abnormalities in LOactivity have been linked to Menkes syndrome and occipital hornsyndrome. Cytosolic forms of the enzyme have been implicated in abnormalcell proliferation (reviewed in Rucker, R. B. et al. (1998) Am. J. Clin.Nutr. 67:996S-1002S and Smith-Mungo, L. I. and H. M. Kagan (1998) MatrixBiol. 16:387-398).

Dihydrofolate Reductases

Dihydrofolate reductases (DHFR) are ubiquitous enzymes that catalyze theNADPH-dependent reduction of dihydrofolate to tetrahydrofolate, anessential step in the de novo synthesis of glycine and purines as wellas the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidinemonophosphate (dTMP). The basic reaction is as follows:7,8-dihydrofolate+NADPH→5,6,7,8-tetrahydrofolate+NADP⁺The enzymes can be inhibited by a number of dihydrofolate analogs,including trimethroprim and methotrexate. Since an abundance of dTMP isrequired for DNA synthesis, rapidly dividing cells require the activityof DHFR. The replication of DNA viruses (i.e., herpesvirus) alsorequires high levels of DHFR activity. As a result, drugs that targetDHFR have been used for cancer chemotherapy and to inhibit DNA virusreplication. (For similar reasons, thymidylate synthetases are alsotarget enzymes.) Drugs that inhibit DHFR are preferentially cytotoxicfor rapidly dividing cells (or DNA virus-infected cells) but have nospecificity, resulting in the indiscriminate destruction of dividingcells. Furthermore, cancer cells may become resistant to drugs such asmethotrexate as a result of acquired transport defects or theduplication of one or more DHFR genes (Stryer, L. (1988) Biochemistry.W.H. Freeman and Co., Inc. New York. pp. 511-519).Aldo/Keto Reductases

Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases withbroad substrate specificities (Bohren, K. M. et al. (1989) J. Biol.Chem. 264:9547-9551). These enzymes catalyze the reduction ofcarbonyl-containing compounds, including carbonyl-containing sugars andaromatic compounds, to the corresponding alcohols. Therefore, a varietyof carbonyl-containing drugs and xenobiotics are likely metabolized byenzymes of this class.

One known reaction catalyzed by a family member, aldose reductase, isthe reduction of glucose to sorbitol, which is then further metabolizedto fructose by sorbitol dehydrogenase. Under normal conditions, thereduction of glucose to sorbitol is a minor pathway. In hyperglycemicstates, however, the accumulation of sorbitol is implicated in thedevelopment of diabetic complications (OMIM #103880 Aldo-keto reductasefamily 1, member B1). Members of this enzyme family are also highlyexpressed in some liver cancers (Cao, D. et al. (1998) J. Biol. Chem.273:11429-11435).

Alcohol Dehydrogenases

Alcohol dehydrogenases (ADHs) oxidize simple alcohols to thecorresponding aldehydes. ADH is a cytosolic enzyme, prefers the cofactorNAD⁺, and also binds zinc ion. Liver contains the highest levels of ADH,with lower levels in kidney, lung, and the gastric mucosa.

Known ADH isoforms are dimeric proteins composed of 40 kDa subunits.There are five known gene loci which encode these subunits (a, b, g, p,c), and some of the loci have characterized allelic variants (b₁, b₂,b₃, g₁, g₂). The subunits can form homodimers and heterodimers; thesubunit composition determines the specific properties of the activeenzyme. The holoenzymes have therefore been categorized as Class I(subunit compositions aa, ab, ag, bg, gg), Class II (pp), and Class III(cc). Class I ADH isozymes oxidize ethanol and other small aliphaticalcohols, and are inhibited by pyrazole. Class II isozymes prefer longerchain aliphatic and aromatic alcohols, are unable to oxidize methanol,and are not inhibited by pyrazole. Class III isozymes prefer even longerchain aliphatic alcohols (five carbons and longer) and aromaticalcohols, and are not inhibited by pyrazole.

The short-chain alcohol dehydrogenases include a number of relatedenzymes with a variety of substrate specificities. Included in thisgroup are the mammalian enzymes D-beta-hydroxybutyrate dehydrogenase,(R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandindehydrogenase, NADPH-dependent carbonyl reductase, corticosteroid11-beta-dehydrogenase, and estradiol 17-beta-dehydrogenase, as well asthe bacterial enzymes acetoacetyl-CoA reductase, glucose1-dehydrogenase, 3-beta-hydroxysteroid dehydrogenase,20-beta-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3-oxoacylreductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase,sorbitol-6-phosphate 2-dehydrogenase, 7-alpha-hydroxysteroiddehydrogenase, cis-1,2-dihydroxy-3,4-cyclohexadiene-1-carboxylatedehydrogenase, cis-toluene dihydrodiol dehydrogenase, cis-benzene glycoldehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase,N-acylmannosamine 1-dehydrogenase, and 2-deoxy-D-gluconate3-dehydrogenase (Krozowski, Z. (1994) J. Steroid Biochem. Mol. Biol.51:125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol. 84:C25-31; andMarks, A. R. et al. (1992) J. Biol. Chem. 267:15459-15463).

Sulfotransferases

Sulfate conjugation occurs on many of the same substrates which undergoO-glucuronidation to produce a highly water-soluble sulfuric acid ester.Sulfotransferases (ST) catalyze this reaction by transferring SO₃ ⁻ fromthe cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to thesubstrate. ST substrates are predominantly phenols and aliphaticalcohols, but also include aromatic amines and aliphatic amines, whichare conjugated to produce the corresponding sulfamates. The products ofthese reactions are excreted mainly in urine.

STs are found in a wide range of tissues, including liver, kidney,intestinal tract, lung, platelets, and brain. The enzymes are generallycytosolic, and multiple forms are often co-expressed. For example, thereare more than a dozen forms of ST in rat liver cytosol. Thesebiochemically characterized STs fall into five classes based on theirsubstrate preference: arylsulfotransferase, alcohol sulfotransferase,estrogen sulfotransferase, tyrosine ester sulfotransferase, and bilesalt sulfotransferase.

ST enzyme activity varies greatly with sex and age in rats. The combinedeffects of developmental cues and sex-related hormones are thought tolead to these differences in ST expression profiles, as well as theprofiles of other DMEs such as cytochromes P450. Notably, the highexpression of STs in cats partially compensates for their low level ofUDP glucuronyltransferase activity.

Several forms of ST have been purified from human liver cytosol andcloned. There are two phenol sulfotransferases with different thermalstabilities and substrate preferences. The thermostable enzyme catalyzesthe sulfation of phenols such as para-nitrophenol, minoxidil, andacetaminophen; the thermolabile enzyme prefers monoamine substrates suchas dopamine, epinephrine, and levadopa. Other cloned STs include anestrogen sulfotransferase and anN-acetylglucosamine-6-O-sulfotransferase. This last enzyme isillustrative of the other major role of STs in cellular biochemistry,the modification of carbohydrate structures that may be important incellular differentiation and maturation of proteoglycans. Indeed, aninherited defect in a sulfotransferase has been implicated in macularcorneal dystrophy, a disorder characterized by a failure to synthesizemature keratan sulfate proteoglycans (Nakazawa, K. et al. (1984) J.Biol. Chem. 259:13751-13757; OMIM #217800 Macular dystrophy, corneal).

Galactosyltransferases

Galactosyltransferases are a subset of glycosyltransferases thattransfer galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc)oligosaccharide chains that are part of glycoproteins or glycolipidsthat are free in solution (Kolbinger, F. et al. (1998) J. Biol. Chem.273:433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473:35-53).Galactosyltransferases have been detected on the cell surface and assoluble extracellular proteins, in addition to being present in theGolgi. β1,3-galactosyltransferases form Type I carbohydrate chains withGal (β1-3)GlcNAc linkages. Known human and mouseβ1,3-galactosyltransferases appear to have a short cytosolic domain, asingle transmembrane domain, and a catalytic domain with eight conservedregions. (Kolbinger et al., supra; and Hennet, T. et al. (1998) J. Biol.Chem. 273:58-65). In mouse UDP-galactose:β-N-acetylglucosamineβ1,3-galactosyltransferase-I region 1 is located at amino acid residues78-83, region 2 is located at amino acid residues 93-102, region 3 islocated at amino acid residues 116-119, region 4 is located at aminoacid residues 147-158, region 5 is located at amino acid residues172-183, region 6 is located at amino acid residues 203-206, region 7 islocated at amino acid residues 236-246, and region 8 is located at aminoacid residues 264-275. A variant of a sequence found within mouseUDP-galactose:β-N-acetylglucosamine β1,3-galactosyltransferase-I region8 is also found in bacterial galactosyltransferases, suggesting thatthis sequence defines a galactosyltransferase sequence motif (Hennet etal., supra). Recent work suggests that brainiac protein is aβ1,3-galactosyltransferase (Yuan, Y. et al. (1997) Cell 88:9-11; andHennet et al., supra).

UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-GalT) (Sato, T. et al.,(1997) EMBO J. 16:1850-1857) catalyzes the formation of Type IIcarbohydrate chains with Gal (β1-4)GlcNAc linkages. As is the case withthe β1,3-galactosyltransferase, a soluble form of the enzyme is formedby cleavage of the membrane-bound form. Amino acids conserved amongβ1,4-galactosyltransferases include two cysteines linked through adisulfide-bond and a putative UDP-galactose-binding site in thecatalytic domain (Yadav, S. and K. Brew (1990) J. Biol. Chem.265:14163-14169; Yadav, S. P. and K. Brew (1991) J. Biol. Chem.266:698-703; and Shaper, N. L. et al. (1997) J. Biol. Chem.272:31389-31399). β1,4-galactosyltransferases have several specializedroles in addition to synthesizing carbohydrate chains on glycoproteinsor glycolipids. In mammals a β1,4-galactosyltransferase, as part of aheterodimer with α-lactalbumin, functions in lactating mammary glandlactose production. A β1,4-galactosyltransferase on the surface of spermfunctions as a receptor that specifically recognizes the egg. Cellsurface β1,4-galactosyltransferases also function in cell adhesion,cell/basal lamina interaction, and normal and metastatic cell migration.(Shur, B. (1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, J. (1995)Adv. Exp. Med. Biol. 376:95-104).

Gamma-glutamyl Transpeptidase

Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes thatinitiate extracellular glutathione (GSH) breakdown by cleavinggamma-glutamyl amide bonds. The breakdown of GSH provides cells with aregional cysteine pool for biosynthetic pathways. Gamma-glutamyltranspeptidases also contribute to cellular antioxidant defenses andexpression is induced by oxidative stress. The cell surface-localizedglycoproteins are expressed at high levels in cancer cells. Studies havesuggested that the high level of gamma-glutamyl transpeptidase activitypresent on the surface of cancer cells could be exploited to activateprecursor drugs, resulting in high local concentrations of anti-cancertherapeutic agents (Hanigan, M. H. (1998) Chem. Biol. Interact.111-112:333-342; Taniguchi, N. and Y. Ikeda (1998) Adv. Enzymol. Relat.Areas Mol. Biol. 72:239-278; Chikhi, N. et al. (1999) Comp. Biochem.Physiol. B. Biochem. Mol. Biol. 122:367-380).

Aminotransferases

Aminotransferases comprise a family of pyridoxal 5′-phosphate(PLP)-dependent enzymes that catalyze transformations of amino acids.Aspartate aminotransferase (AspAT) is the most extensively studiedPLP-containing enzyme. It catalyzes the reversible transamination ofdicarboxylic L-amino acids, aspartate and glutamate, and thecorresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. Other membersof the family include pyruvate aminotransferase, branched-chain aminoacid aminotransferase, tyrosine aminotransferase, aromaticaminotransferase, alanine:glyoxylate aminotransferase (AGT), andkynurenine aminotransferase (Vacca, R. A. et al. (1997) J. Biol. Chem.272:21932-21937).

Primary hyperoxaluria type-1 is an autosomal recessive disorderresulting in a deficiency in the liver-specific peroxisomal enzyme,alanine:glyoxylate aminotransferase-1. The phenotype of the disorder isa deficiency in glyoxylate metabolism. In the absence of AGT, glyoxylateis oxidized to oxalate rather than being transaminated to glycine. Theresult is the deposition of insoluble calcium oxalate in the kidneys andurinary tract, ultimately causing renal failure (Lumb, M. J. et al.(1999) J. Biol. Chem. 274:20587-20596).

Kynurenine aminotransferase catalyzes the irreversible transamination ofthe L-tryptophan metabolite L-kynurenine to form kynurenic acid. Theenzyme may also catalyze the reversible transamination reaction betweenL-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate andL-glutamate. Kynurenic acid is a putative modulator of glutamatergicneurotransmission; thus a deficiency in kynurenine aminotransferase maybe associated with pleotrophic effects (Buchli, R. et al. (1995) J.Biol. Chem. 270:29330-29335).

Catechol-O-methyltransferase

Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methylgroup of S-adenosyl-L-methionine (AdoMet; SAM) donor to one of thehydroxyl groups of the catechol substrate (e.g., L-dopa, dopamine, orDBA). Methylation of the 3′-hydroxyl group is favored over methylationof the 4′-hydroxyl group and the membrane bound isoform of COMT is moreregiospecific than the soluble form. Translation of the soluble form ofthe enzyme results from utilization of an internal start codon in afull-length mRNA (1.5 kb) or from the translation of a shorter mRNA (1.3kb), transcribed from an internal promoter. The proposed S_(N)2-likemethylation reaction requires Mg⁺⁺ and is inhibited by Ca⁺⁺. The bindingof the donor and substrate to COMT occurs sequentially. AdoMet firstbinds COMT in a Mg⁺⁺-independent manner, followed by the binding of Mg⁺⁺and the binding of the catechol substrate.

The amount of COMT in tissues is relatively high compared to the amountof activity normally required, thus inhibition is problematic.Nonetheless, inhibitors have been developed for in vitro use (e.g.,gallates, tropolone, U-0521, and3′,4′-dihydroxy-2-methyl-propiophetropolone) and for clinical use (e.g.,nitrocatechol-based compounds and tolcapone). Administration of theseinhibitors results in the increased half-life of L-dopa and theconsequent formation of dopamine. Inhibition of COMT is also likely toincrease the half-life of various other catechol-structure compounds,including but not limited to epinephrine/norepinephrine, isoprenaline,rimiterol, dobutamine, fenoldopam, apomorphine, and α-methyldopa. Adeficiency in norepinephrine has been linked to clinical depression,hence the use of COMT inhibitors could be useful in the treatment ofdepression. COMT inhibitors are generally well tolerated with minimalside effects and are ultimately metabolized in the liver with only minoraccumulation of metabolites in the body (Männistö, P. T. and S. Kaakkola(1999) Pharmacol. Rev. 51:593-628).

Copper-Zinc Superoxide Dismutases

Copper-zinc superoxide dismutases are compact homodimeric metalloenzymesinvolved in cellular defenses against oxidative damage. The enzymescontain one atom of zinc and one atom of copper per subunit and catalyzethe dismutation of superoxide anions into O₂ and H₂O₂. The rate ofdismutation is diffusion-limited and consequently enhanced by thepresence of favorable electrostatic interactions between the substrateand enzyme active site. Examples of this class of enzyme have beenidentified in the cytoplasm of all the eukaryotic cells as well as inthe periplasm of several bacterial species. Copper-zinc superoxidedismutases are robust enzymes that are highly resistant to proteolyticdigestion and denaturing by urea and SDS. In addition to the compactstructure of the enzymes, the presence of the metal ions andintrasubunit disulfide bonds is believed to be responsible for enzymestability. The enzymes undergo reversible denaturation at temperaturesas high as 70° C. (Battistoni, A. et al. (1998) J. Biol. Chem.273:5655-5661).

Overexpression of superoxide dismutase has been implicated in enhancingfreezing tolerance of transgenic alfalfa as well as providing resistanceto environmental toxins such as the diphenyl ether herbicide,acifluorfen (McKersie, B. D. et al. (1993) Plant Physiol.103:1155-1163). In addition, yeast cells become more resistant tofreeze-thaw damage following exposure to hydrogen peroxide which causesthe yeast cells to adapt to further peroxide stress by upregulatingexpression of superoxide dismutases. In this study, mutations to yeastsuperoxide dismutase genes had a more detrimental effect on freeze-thawresistance than mutations which affected the regulation of glutathionemetabolism, long suspected of being important in determining anorganism's survival through the process of cryopreservation (Jong-InPark, J.-I. et al. (1998) J. Biol. Chem. 273:22921-22928).

Expression of superoxide dismutase is also associated with Mycobacteriumtuberculosis, the organism that causes tuberculosis. Superoxidedismutase is one of the ten major proteins secreted by M. tuberculosisand its expression is upregulated approximately 5-fold in response tooxidative stress. M. tuberculosis expresses almost two orders ofmagnitude more superoxide dismutase than the nonpathogenic mycobacteriumM. smegmatis, and secretes a much higher proportion of the expressedenzyme. The result is the secretion of ˜350-fold more enzyme by M.tuberculosis than M. smegmatis, providing substantial resistance tooxidative stress (Harth, G. and M. A. Horwitz (1999) J. Biol. Chem.274:4281-4292).

The reduced expression of copper-zinc superoxide dismutases, as well asother enzymes with anti-oxidant capabilities, has been implicated in theearly stages of cancer. The expression of copper-zinc superoxidedismutases is reduced in prostatic intraepithelial neoplasia andprostate carcinomas, (Bostwick, D. G. (2000) Cancer 89:123-134).

Phosphoesterases

Phosphotriesterases (PTE, paraoxonases) are enzymes that hydrolyze toxicorganophosphorus compounds and have been isolated from a variety oftissues. Phosphotriesterases play a central role in the detoxificationof insecticides by mammals. Birds and insects lack PTE, and as a resulthave reduced tolerance for organophosphorus compounds (Vilanova, E. andM. A. Sogorb (1999) Crit. Rev. Toxicol. 29:21-57). Phosphotriesteraseactivity varies among individuals and is lower in infants than adults.PTE knockout mice are markedly more sensitive to theorganophosphate-based toxins diazoxon and chlorpyrifos oxon (Furlong, C.E., et al. (2000) Neurotoxicology 21:91-100). Phosphotriesterase is alsoimplicated in atherosclerosis and diseases involving lipoproteinmetabolism.

Glycerophosphoryl diester phosphodiesterase (also known asglycerophosphodiester phosphodiesterase) is a phosphodiesterase whichhydrolyzes deacetylated phospholipid glycerophosphodiesters to producesn-glycerol-3-phosphate and an alcohol. Glycerophosphocholine,glycerophosphoethanolamine, glycerophosphoglycerol, andglycerophosphoinositol are examples of substrates for glycerophosphoryldiester phosphodiesterases. A glycerophosphoryl diesterphosphodiesterase from E. coli has broad specificity forglycerophosphodiester substrates (Larson, T. J. et al. (1983) J. Biol.Chem. 248:5428-5432).

Cyclic nucleotide phosphodiesterases (PDEs) are crucial enzymes in theregulation of the cyclic nucleotides cAMP and cGMP. cAMP and cGMPfunction as intracellular second messengers to transduce a variety ofextracellular signals including hormones, light, and neurotransmitters.PDEs degrade cyclic nucleotides to their corresponding monophosphates,thereby regulating the intracellular concentrations of cyclicnucleotides and their effects on signal transduction. Due to their rolesas regulators of signal transduction, PDEs have been extensively studiedas chemotherapeutic targets (Perry, M. J. and G. A. Higgs (1998) Curr.Opin. Chem. Biol. 2:472-481; Torphy, J. T. (1998) Am. J. Resp. Crit.Care Med. 157:351-370).

Families of mammalian PDEs have been classified based on their substratespecificity and affinity, sensitivity to cofactors, and sensitivity toinhibitory agents (Beavo, J. A. (1995) Physiol. Rev. 75:725-748; Conti,M. et al. (1995) Endocrine Rev. 16:370-389). Several of these familiescontain distinct genes, many of which are expressed in different tissuesas splice variants. Within PDE families, there are multiple isozymes andmultiple splice variants of these isozymes (Conti, M. and S.-L. C. Jin(1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence ofmultiple PDE families, isozymes, and splice variants is an indication ofthe variety and complexity of the regulatory pathways involving cyclicnucleotides (Houslay, M. D. and G. Milligan (1997) Trends Biochem. Sci.22:217-224).

Type 1 PDEs (PDE1s) are Ca²⁺/calmodulin-dependent and appear to beencoded by at least three different genes, each having at least twodifferent splice variants (Kakkar, R. et al. (1999) Cell Mol. Life Sci.55:1164-1186). PDE1s have been found in the lung, heart, and brain. SomePDE1 isozymes are regulated in vitro byphosphorylation/dephosphorylation. Phosphorylation of these PDE1isozymes decreases the affinity of the enzyme for calmodulin, decreasesPDE activity, and increases steady state levels of cAMP (Kakkar et al.,supra). PDE1s may provide useful therapeutic targets for disorders ofthe central nervous system and the cardiovascular and immune systems,due to the involvement of PDE1s in both cyclic nucleotide and calciumsignaling (Perry and Higgs, supra).

PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum,neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle(Sadhu, K. et al. (1999) J. Histochem. Cytochem. 47:895-906). PDE2s arethought to mediate the effects of cAMP on catecholamine secretion,participate in the regulation of aldosterone (Beavo, supra), and play arole in olfactory signal transduction (Juilfs, D. M. et al. (1997) Proc.Natl. Acad. Sci. USA 94:3388-3395).

PDE3s have high affinity for both cGMP and cAMP, and so these cyclicnucleotides act as competitive substrates for PDE3s. PDE3s play roles instimulating myocardial contractility, inhibiting platelet aggregation,relaxing vascular and airway smooth muscle, inhibiting proliferation ofT-lymphocytes and cultured vascular smooth muscle cells, and regulatingcatecholamine-induced release of free fatty acids from adipose tissue.The PDE3 family of phosphodiesterases are sensitive to specificinhibitors such as cilostamide, enoximone, and lixazinone. Isozymes ofPDE3 can be regulated by cAMP-dependent protein kinase, or byinsulin-dependent kinases (Degerman, E. et al. (1997) J. Biol. Chem.272:6823-6826).

PDE4s are specific for cAMP; are localized to airway smooth muscle, thevascular endothelium, and all inflammatory cells; and can be activatedby cAMP-dependent phosphorylation. Since elevation of cAMP levels canlead to suppression of inflammatory cell activation and to relaxation ofbronchial smooth muscle, PDE4s have been studied extensively as possibletargets for novel anti-inflammatory agents, with special emphasis placedon the discovery of asthma treatments. PDE4 inhibitors are currentlyundergoing clinical trials as treatments for asthma, chronic obstructivepulmonary disease, and atopic eczema. All four known isozymes of PDE4are susceptible to the inhibitor rolipram, a compound which has beenshown to improve behavioral memory in mice (Barad, M. et al. (1998)Proc. Natl. Acad. Sci. USA 95:15020-15025). PDE4 inhibitors have alsobeen studied as possible therapeutic agents against acute lung injury,endotoxemia, rheumatoid arthritis, multiple sclerosis, and variousneurological and gastrointestinal indications (Doherty, A. M. (1999)Curr. Opin. Chem. Biol. 3:466-473).

PDE5 is highly selective for cGMP as a substrate (Turko, I. V. et al.(1998) Biochemistry 37:4200-4205), and has two allosteric cGMP-specificbinding sites (McAllister-Lucas, L. M. et al. (1995) J. Biol. Chem.270:30671-30679). Binding of cGMP to these allosteric binding sitesseems to be important for phosphorylation of PDE5 by cGMP-dependentprotein kinase rather than for direct regulation of catalytic activity.High levels of PDE5 are found in vascular smooth muscle, platelets,lung, and kidney. The inhibitor zaprinast is effective against PDE5 andPDE1s. Modification of zaprinast to provide specificity against PDE5 hasresulted in sildenafil (VIAGRA; Pfizer, Inc., New York N.Y.), atreatment for male erectile dysfunction (Terrett, N. et al. (1996)Bioorg. Med. Chem. Lett. 6:1819-1824). Inhibitors of PDE5 are currentlybeing studied as agents for cardiovascular therapy (Perry and Higgs,supra).

PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, arecrucial components of the phototransduction cascade. In association withthe G-protein transducin, PDE6s hydrolyze cGMP to regulate cGMP-gatedcation channels in photoreceptor membranes. In addition to thecGMP-binding active site, PDE6s also have two high-affinity cGMP-bindingsites which are thought to play a regulatory role in PDE6 function(Artemyev, N. O. et al. (1998) Methods 14:93-104). Defects in PDE6s havebeen associated with retinal disease. Retinal degeneration in the rdmouse (Yan, W. et al. (1998) Invest. Opthalmol. Vis. Sci. 39:2529-2536),autosomal recessive retinitis pigmentosa in humans (Danciger, M. et al.(1995) Genomics 30:1-7), and rod/cone dysplasia 1 in Irish Setter dogs(Suber, M. L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972)have been attributed to mutations in the PDE6B gene.

The PDE7 family of PDEs consists of only one known member havingmultiple splice variants (Bloom, T. J. and J. A. Beavo (1996) Proc.Natl. Acad. Sci. USA 93:14188-14192). PDE7s are cAMP specific, butlittle else is known about their physiological function. Although mRNAsencoding PDE7s are found in skeletal muscle, heart, brain, lung, kidney,and pancreas, expression of PDE7 proteins is restricted to specifictissue types (Han, P. et al. (1997) J. Biol. Chem. 272:16152-16157;Perry and Higgs, supra). PDE7s are very closely related to the PDE4family; however, PDE7s are not inhibited by rolipram, a specificinhibitor of PDE4s (Beavo, supra).

PDE8s are cAMP specific, and are closely related to the PDE4 family.PDE8s are expressed in thyroid gland, testis, eye, liver, skeletalmuscle, heart, kidney, ovary, and brain. The cAMP-hydrolyzing activityof PDE8s is not inhibited by the PDE inhibitors rolipram, vinpocetine,milrinone, IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDE8sare inhibited by dipyridamole (Fisher, D. A. et al. (1998) Biochem.Biophys. Res. Commun. 246:570-577; Hayashi, M. et al. (1998) Biochem.Biophys. Res. Commun. 250:751-756; Soderling, S. H. et al. (1998) Proc.Natl. Acad. Sci. USA 95:8991-8996).

PDE9s are cGMP specific and most closely resemble the PDE8 family ofPDEs. PDE9s are expressed in kidney, liver, lung, brain, spleen, andsmall intestine. PDE9s are not inhibited by sildenafil (VIAGRA; Pfizer,Inc., New York N.Y.), rolipram, vinpocetine, dipyridamole, or IBMX(3-isobutyl-1-methylxanthine), but they are sensitive to the PDE5inhibitor zaprinast (Fisher, D. A. et al. (1998) J. Biol. Chem.273:15559-15564; Soderling, S. H. et al. (1998) J. Biol. Chem.273:15553-15558).

PDE10s are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDE10sare expressed in brain, thyroid, and testis. (Soderling, S. H. et al.(1999) Proc. Natl. Acad. Sci. USA 96:7071-7076; Fujishige, K. et al.(1999) J. Biol. Chem. 274:18438-18445; Loughney, K. et al (1999) Gene234:109-117).

PDEs are composed of a catalytic domain of about 270-300 amino acids, anN-terminal regulatory domain responsible for binding cofactors, and, insome cases, a hydrophilic C-terminal domain of unknown function (Contiand Jin, supra). A conserved, putative zinc-binding motif has beenidentified in the catalytic domain of all PDEs. N-terminal regulatorydomains include non-catalytic cGMP-binding domains in PDE2s, PDE5s, andPDE6s; calmodulin-binding domains in PDE1s; and domains containingphosphorylation sites in PDE3s and PDE4s. In PDE5, the N-terminalcGMP-binding domain spans about 380 amino acid residues and comprisestandem repeats of a conserved sequence motif (McAllister-Lucas, L. M. etal. (1993) J. Biol. Chem. 268:22863-22873). The NKXnD motif has beenshown by mutagenesis to be important for cGMP binding (Turko, I. V. etal. (1996) J. Biol. Chem. 271:22240-22244). PDE families displayapproximately 30% amino acid identity within the catalytic domain;however, isozymes within the same family typically display about 85-95%identity in this region (e.g. PDE4A vs PDE4B). Furthermore, within afamily there is extensive similarity (>60%) outside the catalyticdomain; while across families, there is little or no sequence similarityoutside this domain.

Many of the constituent functions of immune and inflammatory responsesare inhibited by agents that increase intracellular levels of cAMP(Verghese, M. W. et al. (1995) Mol. Pharmacol. 47:1164-1171). A varietyof diseases have been attributed to increased PDE activity andassociated with decreased levels of cyclic nucleotides. For example, aform of diabetes insipidus in mice has been associated with increasedPDE4 activity, an increase in low-K_(m) cAMP PDE activity has beenreported in leukocytes of atopic patients, and PDE3 has been associatedwith cardiac disease.

Many inhibitors of PDEs have undergone clinical evaluation (Perry andHiggs, supra; Torphy, T. J. (1998) Am. J. Respir. Crit. Care Med.157:351-370). PDE3 inhibitors are being developed as antithromboticagents, antihypertensive agents, and as cardiotonic agents useful in thetreatment of congestive heart failure. Rolipram, a PDE4 inhibitor, hasbeen used in the treatment of depression, and other PDE4 inhibitors havean anti-inflammatory effect. Rolipram may inhibit HIV-1 replication(Angel, J. B. et al. (1995) AIDS 9:1137-1144). Additionally, rolipramsuppresses the production of cytokines such as TNF-a and b andinterferon g, and thus is effective against encephalomyelitis. Roliprammay also be effective in treating tardive dyskinesia and multiplesclerosis (Sommer, N. et al. (1995) Nat. Med. 1:244-248; Sasaki, H. etal. (1995) Eur. J. Pharmacol. 282:71-76). Theophylline is a nonspecificPDE inhibitor used in treatment of bronchial asthma and otherrespiratory diseases. Theophylline is believed to act on airway smoothmuscle function and in an anti-inflammatory or immunomodulatory capacityBanner, K. H. and C. P. Page (1995) Eur. Respir. J. 8:996-1000).Pentoxifylline is another nonspecific PDE inhibitor used in thetreatment of intermittent claudication and diabetes-induced peripheralvascular disease. Pentoxifylline is also known to block TNF-a productionand may inhibit HIV-1 replication (Angel et al., supra).

PDEs have been reported to affect cellular proliferation of a variety ofcell types (Conti et al. (1995) Endocrine Rev. 16:370-389) and have beenimplicated in various cancers. Growth of prostate carcinoma cell linesDU145 and LNCaP was inhibited by delivery of cAMP derivatives and PDEinhibitors (Bang, Y. J. et al. (1994) Proc. Natl. Acad. Sci. USA91:5330-5334). These cells also showed a permanent conversion inphenotype from epithelial to neuronal morphology. It has also beensuggested that PDE inhibitors can regulate mesangial cell proliferation(Matousovic, K. et al. (1995) J. Clin. Invest. 96:401-410) andlymphocyte proliferation (Joulain, C. et al. (1995) J. Lipid Mediat.Cell Signal. 11:63-79). One cancer treatment involves intracellulardelivery of PDEs to particular cellular compartments of tumors,resulting in cell death (Deonarain, M. P. and A. A. Epenetos (1994) Br.J. Cancer 70:786-794).

Members of the UDP glucuronyltransferase family (UGTs) catalyze thetransfer of a glucuronic acid group from the cofactor uridinediphosphate-glucuronic acid (UDP-glucuronic acid) to a substrate. Thetransfer is generally to a nucleophilic heteroatom (O, N, or S).Substrates include xenobiotics which have been functionalized by Phase Ireactions, as well as endogenous compounds such as bilirubin, steroidhormones, and thyroid hormones. Products of glucuronidation are excretedin urine if the molecular weight of the substrate is less than about 250g/mol, whereas larger glucuronidated substrates are excreted in bile.

UGTs are located in the microsomes of liver, kidney, intestine, skin,brain, spleen, and nasal mucosa, where they are on the same side of theendoplasmic reticulum membrane as cytochrome P450 enzymes andflavin-containing monooxygenases. UGTs have a C-terminalmembrane-spanning domain which anchors them in the endoplasmic reticulummembrane, and a conserved signature domain of about 50 amino acidresidues in their C terminal section (PROSITE PDOC00359UDP-glycosyltransferase signature).

UGTs involved in drug metabolism are encoded by two gene families, UGT1and UGT2. Members of the UGT1 family result from alternative splicing ofa single gene locus, which has a variable substrate binding domain andconstant region involved in cofactor binding and membrane insertion.Members of the UGT2 family are encoded by separate gene loci, and aredivided into two families, UGT2A and UGT2B. The 2A subfamily isexpressed in olfactory epithelium, and the 2B subfamily is expressed inliver microsomes. Mutations in UGT genes are associated withhyperbilirubinemia (OMIM #143500 Hyperbilirubinemia I); Crigler-Najjarsyndrome, characterized by intense hyperbilirubinemia from birth (OMIM#218800 Crigler-Najjar syndrome); and a milder form ofhyperbilirubinemia termed Gilbert's disease (OMIM #191740 UGT1).

Thioesterases

Two soluble thioesterases involved in fatty acid biosynthesis have beenisolated from mammalian tissues, one which is active only towardlong-chain fatty-acyl thioesters and one which is active towardthioesters with a wide range of fatty-acyl chain-lengths. Thesethioesterases catalyze the chain-terminating step in the de novobiosynthesis of fatty acids. Chain termination involves the hydrolysisof the thioester bond which links the fatty acyl chain to the4′-phosphopantetheine prosthetic group of the acyl carrier protein (ACP)subunit of the fatty acid synthase (Smith, S. (1981a) Methods Enzymol.71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).

E. coli contains two soluble thioesterases, thioesterase I which isactive only toward long-chain acyl thioesters, and thioesterase II(TEII) which has a broad chain-length specificity (Naggert, J. et al.(1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not exhibitsequence similarity with either of the two types of mammalianthioesterases which function as chain-terminating enzymes in de novofatty acid biosynthesis. Unlike the mammalian thioesterases, E. coliTEII lacks the characteristic serine active site gly-X-ser-X-glysequence motif and is not inactivated by the serine modifying agentdiisopropyl fluorophosphate. However, modification of histidine 58 byiodoacetamide and diethylpyrocarbonate abolished TEII activity.Overexpression of TEII did not alter fatty acid content in E. coli,which suggests that it does not function as a chain-terminating enzymein fatty acid biosynthesis (Naggert et al., supra). For that reason,Naggert et al. (supra) proposed that the physiological substrates for E.coli TEII may be coenzyme A (CoA)-fatty acid esters instead ofACP-phosphopanthetheine-fatty acid esters.

Carboxylesterases

Mammalian carboxylesterases are a multigene family expressed in avariety of tissues and cell types. Acetylcholinesterase,butyrylcholinesterase, and carboxylesterase are grouped into the serinesuperfamily of esterases (B-esterases). Other carboxylesterases includethyroglobulin, thrombin, Factor IX, gliotactin, and plasminogen.Carboxylesterases catalyze the hydrolysis of ester- and amide-groupsfrom molecules and are involved in detoxification of drugs,environmental toxins, and carcinogens. Substrates for carboxylesterasesinclude short- and long-chain acyl-glycerols, acylcarnitine, carbonates,dipivefrin hydrochloride, cocaine, salicylates, capsaicin,palmitoyl-coenzyme A, imidapril, haloperidol, pyrrolizidine alkaloids,steroids, p-nitrophenyl acetate, malathion, butanilicaine, andisocarboxazide. Carboxylesterases are also important for the conversionof prodrugs to free acids, which may be the active form of the drug(e.g., lovastatin, used to lower blood cholesterol) (reviewed in Satoh,T. and Hosokawa, M. (1998) Annu. Rev. Pharmacol. Toxicol. 38:257-288).Neuroligins are a class of molecules that (i) have N-terminal signalsequences, (ii) resemble cell-surface receptors, (iii) containcarboxylesterase domains, (iv) are highly expressed in the brain, and(v) bind to neurexins in a calcium-dependent manner. Despite thehomology to carboxylesterases, neuroligins lack the active site serineresidue, implying a role in substrate binding rather than catalysis(Ichtchenko, K. et al. (1996) J. Biol. Chem. 271:2676-2682).

Squalene Epoxidase

Squalene epoxidase (squalene monooxygenase, SE) is a microsomalmembrane-bound, FAD-dependent oxidoreductase that catalyzes the firstoxygenation step in the sterol biosynthetic pathway of eukaryotic cells.Cholesterol is an essential structural component of cytoplasmicmembranes acquired via the LDL receptor-mediated pathway or thebiosynthetic pathway. SE converts squalene to 2,3(S)oxidosqualene, whichis then converted to lanosterol and then cholesterol.

High serum cholesterol levels result in the formation of atheroscleroticplaques in the arteries of higher organisms. This deposition of highlyinsoluble lipid material onto the walls of essential blood vesselsresults in decreased blood flow and potential necrosis. HMG-CoAreductase is responsible for the first committed step in cholesterolbiosynthesis, conversion of 3-hydroxyl-3-methyl-glutaryl CoA (HMG-CoA)to mevalonate. HMG-CoA is the target of a number of pharmaceuticalcompounds designed to lower plasma cholesterol levels, but inhibition ofMSG-CoA also results in the reduced synthesis of non-sterolintermediates required for other biochemical pathways. Since SEcatalyzes a rate-limiting reaction that occurs later in the sterolsynthesis pathway with cholesterol as the only end product, SE is abetter ideal target for the design of anti-hyperlipidemic drugs(Nakamura, Y. et al. (1996) 271:8053-8056).

Epoxide Hydrolases

Epoxide hydrolases catalyze the addition of water to epoxide-containingcompounds, thereby hydrolyzing epoxides to their corresponding1,2-diols. They are related to bacterial haloalkane dehalogenases andshow sequence similarity to other members of the α/β hydrolase foldfamily of enzymes. This family of enzymes is important for thedetoxification of xenobiotic epoxide compounds which are often highlyelectrophilic and destructive when introduced. Examples of epoxidehydrolase reactions include the hydrolysis of some leukotoxin toleukotoxin diol, and isoleukotoxin to isoleukotoxin diol. Leukotoxinsalter membrane permeability and ion transport and cause inflammatoryresponses. In addition, epoxide carcinogens are produced by cytochromeP450 as intermediates in the detoxification of drugs and environmentaltoxins. Epoxide hydrolases possess a catalytic triad composed of Asp,Asp, and His (Arand, M. et al. (1996) J. Biol. Chem. 271:4223-4229;Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657; Argiriadi, M. A.et al. (2000) J. Biol. Chem. 275:15265-15270).

Enzymes Involved in Tyrosine Catalysis

The degradation of the amino acid tyrosine, to either succinate andpyruvate or fumarate and acetoacetate, requires a large number ofenzymes and generates a large number of intermediate compounds. Inaddition, many xenobiotic compounds may be metabolized using one or morereactions that are part of the tyrosine catabolic pathway. Enzymesinvolved in the degradation of tyrosine to succinate and pyruvate (e.g.,in Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase,4-hydroxyphenylacetate 3-hydroxylase, 3,4-dihydroxyphenylacetate2,3-dioxygenase, 5-carboxymethyl-2-hydroxymuconic semialdehydedehydrogenase, trans,cis-5-carboxymethyl-2-hydroxymuconate isomerase,homoprotocatechuate isomerase/decarboxylase,cis-2-oxohept-3-ene-1,7-dioate hydratase,2,4-dihydroxyhept-trans-2-ene-1,7-dioate aldolase, and succinicsemialdehyde dehydrogenase. Enzymes involved in the degradation oftyrosine to fumarate and acetoacetate (e.g., in Pseudomonas species)include 4-hydroxyphenylpyruvate dioxygenase, homogentisate1,2-dioxygenase, maleylacetoacetate isomerase, fumarylacetoacetase and4-hydroxyphenylacetate. Additional enzymes associated with tyrosinemetabolism in different organisms include4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase,5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase,2-oxo-hept-3-ene-1,7-dioate hydratase, and5-carboxymethyl-2-hydroxymuconate isomerase (Ellis, L. B. M. et al.(1999) Nucleic Acids Res. 27:373-376; Wackett, L. P. and Ellis, L. B. M.(1996) J. Microbiol. Meth. 25:91-93; and Schmidt, M. (1996) Amer. Soc.Microbiol. News 62:102).

In humans, acquired or inherited genetic defects in enzymes of thetyrosine degradation pathway may result in hereditary tyrosinemia. Oneform of this disease, hereditary tyrosinemia 1 (HT1) is caused by adeficiency in the enzyme fumarylacetoacetate hydrolase, the last enzymein the pathway in organisms that metabolize tyrosine to fumarate andacetoacetate. HT1 is characterized by progressive liver damage beginningat infancy, and increased risk for liver cancer (Endo, F. et al. (1997)J. Biol. Chem. 272:24426-24432).

Expression Profiling

Microarrays are analytical tools used in bioanalysis. A microarray has aplurality of molecules spatially distributed over, and stably associatedwith, the surface of a solid support. Microarrays of polypeptides,polynucleotides, and/or antibodies have been developed and find use in avariety of applications, such as gene sequencing, monitoring geneexpression, gene mapping, bacterial identification, drug discovery, andcombinatorial chemistry.

One area in particular in which microarrays find use is in geneexpression analysis. Array technology can provide a simple way toexplore the expression of a single polymorphic gene or the expressionprofile of a large number of related or unrelated genes. When theexpression of a single gene is examined, arrays are employed to detectthe expression of a specific gene or its variants. When an expressionprofile is examined, arrays provide a platform for identifying genesthat are tissue specific, are affected by a substance being tested in atoxicology assay, are part of a signaling cascade, carry outhousekeeping functions, or are specifically related to a particulargenetic predisposition, condition, disease, or disorder.

Expression Information

DNA methylation is an epigenetic process that alters gene expression inmammalian cells. Methylation of cytosine residues occurs at specific5′-CG-3′ dinucleotide base pairs during DNA replication. A high densityof CG dinucleotides, termed CpG islands (CGI), are found near thepromoters of approximately 60% of human genes. Methylation of CGI isusually associated with decreased gene expression (methylationsilencing), presumably by interfering with transcription factor bindingat the promoter. The compound 5-aza-2-deoxycytidine (5-aza-DC) is anirreversible inhibitor of DNA methytransferase that has been commonlyused to demethylate DNA and restore expression of methylation silencedgenes. Methylation of many genes occurs normally during development aspart of X chromosome inactivation and genomic imprinting, and aprogressive increase in gene methylation is associated with aging.

Abnormal DNA methylation including global hypomethylation and regionalhypermethylation is a common feature of human neoplasms and has recentlybeen identified as an important pathway in tumor progression. A cancerspecific methylation pattern, termed “CpG island methylation phenotype”(CIMP) has been described in a distinct subset of colorectal primarytumors and cell lines. CIMP is distinct from the pattern of genemethylation seen in association with aging in non-tumorous colorectaltissues (Toyota et al. 2000; PNAS 97:710-715). Recently,hypermethylation has emerged as a significant mechanism of tumorsuppressor gene inactivation in cancer. For example, methylationsilencing of a key mismatch repair enzyme, hMLH1, has been implicated asa cause of microsatellite instability (MSI), a form of geneticinstability commonly seen in colorectal cancer (CRC) (Herman et al.(1998) Proc Natl Acad Sci 95:6870-6875). Other tumor suppressor genesshown to be targets of methylation silencing in cancer includep16^(INK4a), VHL, BRCA1, TIMP-3, ER, and E-cadherin (Baylin and Herman(2000) Trends Genet 16:168-174).

Colorectal cancer is the fourth most common cancer and the second mostcommon cause of cancer death in the United States with approximately130,000 new cases and 55,000 deaths per year. CRC progresses slowly frombenign adenomatous polyps to invasive metastatic carcinomas. As withother cancer types, tumor progression involves various forms of genomicinstability such as chromosome loss and deletions, MSI, and mutations inkey tumor suppressor genes and proto-oncogenes. For example,approximately 85% of all CRC cases involve an inactivating mutation inthe tumor suppressor gene APC and this is the earliest known geneticevent leading to tumor initiation. During tumor progression, most CRCsacquire additional mutations in other tumor suppressors andproto-oncogenes including K-ras, p53, DCC, TGFbRII, and BAX. The vastmajority of CRCs are sporadic, however two genetic syndromes thatinvolve a high predisposition to CRC include familial adenomatouspolyposis coli (FAP) and hereditary nonpolyposis coli (HNPCC ). FAP iscaused by germline inheritance of an inactivating mutation in APC thatleads to a very high frequency of polyp formation, some of whichprogress to malignant carcinoma. HNPCC is associated with a germlinemutation in the DNA mismatch repair enzymes hMLH1 or hMSH2.

In the APC deficient “MIN” mouse model of colorectal cancer, 5-aza-DCtreatment in combination with a genetic reduction in DNAmethyltransferase I activity leads to reduced polyp formation. Thissuggests that methylation silencing may play a significant role in polypformation in colorectal cancer and that 5-Aza-DC treatment may bebeneficial (Laird et al. 1995; Cell 81:197-205). Using a combination ofmicroarray experiments and other methods, Karpf et al. (1999; Proc NatlAcad Sci USA 96:14007-14012) showed that treatment of cultured HT-29cells, a colorectal cancer cell line, with 5-aza-DC leads to specificexpression of several genes related to interferon (IFN) signaling. Inaddition, 5-aza-DC treatment inhibits growth of HT-29 cells in cultureand this inhibition parallels induction of IFN responsive genes,consistent with the known growth inhibitory function of IFN (Karpf etal., supra). Thus, activation of methylation silenced genes such asgenes associated with IFN signaling may improve growth control in tumorcells.

Array technology can provide a simple way to explore the expression of asingle polymorphic gene or the expression profile of a large number ofrelated or unrelated genes. When the expression of a single gene isexamined, arrays are employed to detect the expression of a specificgene or its variants. When an expression profile is examined, arraysprovide a platform for examining which genes are tissue specific,carrying out housekeeping functions, parts of a signaling cascade, orspecifically related to a particular genetic predisposition, condition,disease, or disorder. The potential application of gene expressionprofiling is particularly relevant to improving diagnosis, prognosis,and treatment of disease. For example, both the levels and sequencesexpressed in tissues from subjects with colon cancer may be comparedwith the levels and sequences expressed in normal tissue.

The present invention provides for a combination comprising a pluralityof cDNAs for use in detecting changes in expression of genes encodingproteins that are associated with DNA methylation. Such a combinationcan be employed for the diagnosis, prognosis or treatment of cancerscorrelated with differential gene expression. The present inventionsatisfies a need in the art by providing a set of differentiallyexpressed genes which may be used entirely or in part to diagnose, tostage, to treat, or to monitor the progression or treatment of a subjectwith a disorder such as colorectal cancer.

Staphylococcal exotoxins specifically activate human T cells, expressingan appropriate TCR-Vbeta chain. Although polyclonal in nature, T cellsactivated by Staphylococcal exotoxins require antigen presenting cells(APCs) to present the exotoxin molecules to the T cells and deliver thecostimulatory signals required for optimum T cell activation. AlthoughStaphylococcal exotoxins must be presented to T cells by APCs, thesemolecules need not be processed by APC. Staphylococcal exotoxinsdirectly bind to a non-polymorphic portion of the human MHC class IImolecules.

Adipose tissue stores and releases fat. Adipose tissue is also one ofthe important target tissues for insulin. Adipogenesis and insulinresistance in type II diabetes are linked. Most patients with type IIdiabetes are obese, and obesity in turn causes insulin resistance.Thiazolidinediones, or peroxisome proliferator-activated receptor gammaagonists (PPAR-γ agonists), are a new class of antidiabetic agents thatimprove insulin sensitivity and reduce plasma glucose and blood pressurein patients with type II diabetes. These agents can bind and activate anorphan nuclear receptor, peroxisome proliferator-activated receptorgamma (PPAR-γ). Thiazolidinediones, a family of PPAR agonist drugs thatincrease sensitivity to insulin, induce preadipocytes to differentiateinto mature fat cells.

Colon Cancer

While soft tissue sarcomas are relatively rare, more than 50% of newpatients diagnosed with the disease will die from it. The molecularpathways leading to the development of sarcomas are relatively unknown,due to the rarity of the disease and variation in pathology. Coloncancer evolves through a multi-step process whereby pre-malignantcolonocytes undergo a relatively defined sequence of events leading totumor formation. Several factors participate in the process of tumorprogression and malignant transformation including genetic factors,mutations, and selection.

To understand the nature of gene alterations in colorectal cancer, anumber of studies have focused on the inherited syndromes. Familialadenomatous polyposis (FAP), is caused by mutations in the adenomatouspolyposis coli gene (APC), resulting in truncated or inactive forms ofthe protein. This tumor suppressor gene has been mapped to chromosome5q. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused bymutations in mis-match repair genes. Although hereditary colon cancersyndromes occur in a small percentage of the population and mostcolorectal cancers are considered sporadic, knowledge from studies ofthe hereditary syndromes can be generally applied. For instance, somaticmutations in APC occur in at least 80% of sporadic colon tumors. APCmutations are thought to be the initiating event in the disease. Othermutations occur subsequently. Approximately 50% of colorectal cancerscontain activating mutations in ras, while 85% contain inactivatingmutations in p53. Changes in all of these genes lead to gene expressionchanges in colon cancer.

C3A Cells

The human C3A cell line is a clonal derivative of HepG2/C3 (hepatomacell line, isolated from a 15-year-old male with liver tumor), which wasselected for strong contact inhibition of growth. The use of a clonalpopulation enhances the reproducibility of the cells. C3A cells havemany characteristics of primary human hepatocytes in culture: i)expression of insulin receptor and insulin-like growth factor IIreceptor; ii) secretion of a high ratio of serum albumin compared withα-fetoprotein; iii) conversion of ammonia to urea and glutamine; iv)metabolism of aromatic amino acids; and v) proliferation in glucose-freeand insulin-free medium. The C3A cell line is now well established as anin vitro model of the mature human liver (Mickelson et al. (1995)Hepatology 22:866-875; Nagendra et al. (1997) Am. J. Physiol.272:G408-G416).

Gemfibrozil is a fibric acid antilipemic agent that lowers serumtriglycerides and produces favorable changes in lipoproteins.Gemfibrozil is effective in reducing the risk of coronary heart diseasein men (Frick, M. H., et al. (1987) New Engl. J. Med. 317:1237-1245).The compound can inhibit peripheral lipolysis and decrease hepaticextraction of free fatty acids, which decreases hepatic triglycerideproduction. Gemfibrozil also inhibits the synthesis and increases theclearance of apolipoprotein B, a carrier molecule for VLDL. Gemfibrozilhas variable effects on LDL cholesterol. Although it causes moderatereductions in patients with type IIa hyperlipoproteinemia, changes inpatients with either type IIb or type IV hyperlipoproteinemia areunpredictable. In general, the HMG-CoA reductase inhibitors are moreeffective than gemfibrozil in reducing LDL cholesterol. At the molecularlevel gemfibozil may function as a peroxisome proliferator-activatedreceptor (PPAR) agonist. Gemfibrozil is rapidly and completely absorbedfrom the GI tract and undergoes enterohepatic recirculation. Gemfibrozilis metabolized by the liver and excreted by the kidneys, mainly asmetabolites, one of which possesses pharmacologic activity. Gemfibozilcauses peroxisome proliferation and hepatocarcinogenesis in rats, whichis a cause for concern generally for fibric acid derivative drugs. Inhumans, fibric acid derivatives are known to increase the risk of gallbladder disease although gemfibrozil is better tolerated than otherfibrates. The relative safety of gemfibrozil in humans compared torodent species including rats may be attributed to differences inmetabolism and clearance of the compound in different species (Dix, K.J., et al. (1999) Drug Metab. Distrib. 27:138-146; Thomas, B. F., et al.(1999) Drug Metab. Distrib. 27:147-157).

There is a need in the art for new compositions, including nucleic acidsand proteins, for the diagnosis, prevention, and treatment ofautoimmune/inflammatory disorders, infectious disorders, immunedeficiencies, disorders of metabolism, reproductive disorders,neurological disorders, cardiovascular disorders, eye disorders, andcell proliferative disorders, including cancer.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides,enzymes, referred to collectively as ‘ENZM’ and individually as‘ENZM-1,’ ‘ENZM-2,’ ‘ENZM-3,’ ‘ENZM-4,’ ‘ENZM-5,’ ‘ENZM-6,’ ‘ENZM-7,’‘ENZM-8,’ ‘ENZM-9,’ ‘ENZM-10,’ ‘ENZM-11,’ ‘ENZM-12,’ ‘ENZM-13,’‘ENZM-14,’ ‘ENZM-15,’ ‘ENZM-16,’ ‘ENZM-17,’ ‘ENZM-18,’ ‘ENZM-19,’‘ENZM-20,’ ‘ENZM-21,’ ‘ENZM-22,’ ‘ENZM-23,’ ‘ENZM-24,’ ‘ENZM-25,’‘ENZM-26,’ ‘ENZM-27,’ ‘ENZM-28,’ ‘ENZM-29,’ ‘ENZM-30,’ ‘ENZM-31,’‘ENZM-32,’ ‘ENZM-33,’ ‘ENZM-34,’ ‘ENZM-35,’ ‘ENZM-36,’ ‘ENZM-37,’‘ENZM-38,’ ‘ENZM-39,’ ‘ENZM-40,’ ‘ENZM-41,’ ‘ENZM-42,’ ‘ENZM-43,’‘ENZM-44,’ ‘ENZM-45,’ ‘ENZM-46,’ ‘ENZM-47,’ ‘ENZM-48,’ ‘ENZM-49,’‘ENZM-50,’ ‘ENZM-51,’ ‘ENZM-52,’ and ‘ENZM-53’ and methods for usingthese proteins and their encoding polynucleotides for the detection,diagnosis, and treatment of diseases and medical conditions. Embodimentsalso provide methods for utilizing the purified enzymes and/or theirencoding polynucleotides for facilitating the drug discovery process,including determination of efficacy, dosage, toxicity, and pharmacology.Related embodiments provide methods for utilizing the purified enzymesand/or their encoding polynucleotides for investigating the pathogenesisof diseases and medical conditions.

An embodiment provides an isolated polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-53, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-53. Another embodiment provides anisolated polypeptide comprising an amino acid sequence of SEQ IDNO:1-53.

Still another embodiment provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-53, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-53, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-53, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-53. Inanother embodiment, the polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO:1-53. In an alternativeembodiment, the polynucleotide is selected from the group consisting ofSEQ ID NO:54-106.

Still another embodiment provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53. Another embodiment provides a celltransformed with the recombinant polynucleotide. Yet another embodimentprovides a transgenic organism comprising the recombinantpolynucleotide.

Another embodiment provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-53, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical or at least about 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-53, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-53, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-53. Themethod comprises a) culturing a cell under conditions suitable forexpression of the polypeptide, wherein said cell is transformed with arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide encoding the polypeptide, and b) recoveringthe polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specificallybinds to a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53.

Still yet another embodiment provides an isolated polynucleotideselected from the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:54-106, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:54-106, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In otherembodiments, the polynucleotide can comprise at least about 20, 30, 40,60, 80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:54-106, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:54-106, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) hybridizing the sample with a probe comprising at least 20contiguous nucleotides comprising a sequence complementary to saidtarget polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex. In a related embodiment, themethod can include detecting the amount of the hybridization complex. Instill other embodiments, the probe can comprise at least about 20, 30,40, 60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:54-106, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:54-106, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) amplifying said target polynucleotide or fragment thereofusing polymerase chain reaction amplification, and b) detecting thepresence or absence of said amplified target polynucleotide or fragmentthereof. In a related embodiment, the method can include detecting theamount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amountof a polypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-53, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-53, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-53, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-53, anda pharmaceutically acceptable excipient. In one embodiment, thecomposition can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53. Other embodiments provide a method oftreating a disease or condition associated with decreased or abnormalexpression of functional ENZM, comprising administering to a patient inneed of such treatment the composition.

Yet another embodiment provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-53, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-53. The method comprises a) exposinga sample comprising the polypeptide to a compound, and b) detectingagonist activity in the sample. Another embodiment provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. Yet another embodiment providesa method of treating a disease or condition associated with decreasedexpression of functional ENZM, comprising administering to a patient inneed of such treatment the composition.

Still yet another embodiment provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-53, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-53. The method comprises a) exposinga sample comprising the polypeptide to a compound, and b) detectingantagonist activity in the sample. Another embodiment provides acomposition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. Yet another embodimentprovides a method of treating a disease or condition associated withoverexpression of functional ENZM, comprising administering to a patientin need of such treatment the composition.

Another embodiment provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-53, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical or atleast about 90% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-53, c) a biologically active fragment ofa polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53. The method comprises a) combining thepolypeptide with at least one test compound under suitable conditions,and b) detecting binding of the polypeptide to the test compound,thereby identifying a compound that specifically binds to thepolypeptide.

Yet another embodiment provides a method of screening for a compoundthat modulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-53, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-53, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-53. The method comprises a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Still yet another embodiment provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:54-106, the methodcomprising a) exposing a sample comprising the target polynucleotide toa compound, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.

Another embodiment provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:54-106, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical or at least about 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:54-106, iii) a polynucleotide having a sequence complementary to i),iv) a polynucleotide complementary to the polynucleotide of ii), and v)an RNA equivalent of i)-iv). Hybridization occurs under conditionswhereby a specific hybridization complex is formed between said probeand a target polynucleotide in the biological sample, said targetpolynucleotide selected from the group consisting of i) a polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:54-106, ii) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical or at leastabout 90% identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:54-106, iii) a polynucleotide complementary tothe polynucleotide of i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv).Alternatively, the target polynucleotide can comprise a fragment of apolynucleotide selected from the group consisting of i)-v) above; c)quantifying the amount of hybridization complex; and d) comparing theamount of hybridization complex in the treated biological sample withthe amount of hybridization complex in an untreated biological sample,wherein a difference in the amount of hybridization complex in thetreated biological sample is indicative of toxicity of the testcompound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide andpolypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog, and the PROTEOME database identificationnumbers and annotations of PROTEOME database homologs, for polypeptideembodiments of the invention. The probability scores for the matchesbetween each polypeptide and its homolog(s) are also shown.

Table 3 shows structural features of polypeptide embodiments, includingpredicted motifs and domains, along with the methods, algorithms, andsearchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide embodiments, along with selected fragments ofthe polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotideembodiments.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyzepolynucleotides and polypeptides, along with applicable descriptions,references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotidesequences of the invention, along with allele frequencies in differenthuman populations.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described,it is understood that embodiments of the invention are not limited tothe particular machines, instruments, materials, and methods described,as these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a host cell” includes aplurality of such host cells, and a reference to “an antibody” is areference to one or more antibodies and equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with variousembodiments of the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

“ENZM” refers to the amino acid sequences of substantially purified ENZMobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of ENZM. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of ENZM either by directlyinteracting with ENZM or by acting on components of the biologicalpathway in which ENZM participates.

An “allelic variant” is an alternative form of the gene encoding ENZM.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding ENZM include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as ENZM or a polypeptide with atleast one functional characteristic of ENZM. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingENZM, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotideencoding ENZM. The encoded protein may also be “altered,” and maycontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentENZM. Deliberate amino acid substitutions may be made on the basis ofone or more similarities in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of ENZM isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valine; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” can refer to anoligopeptide, a peptide, a polypeptide, or a protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. Where “amino acid sequence” is recited to refer to a sequenceof a naturally occurring protein molecule, “amino acid sequence” andlike terms are not meant to limit the amino acid sequence to thecomplete native amino acid sequence associated with the recited proteinmolecule.

“Amplification” relates to the production of additional copies of anucleic acid. Amplification may be carried out using polymerase chainreaction (PCR) technologies or other nucleic acid amplificationtechnologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of ENZM. Antagonists may include proteins suchas antibodies, anticalins, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition which modulates theactivity of ENZM either by directly interacting with ENZM or by actingon components of the biological pathway in which ENZM participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind ENZMpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KYH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a polynucleotide having a specificnucleic acid sequence. Antisense compositions may include DNA; RNA;peptide nucleic acid (PNA); oligonucleotides having modified backbonelinkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oligonucleotides having modified sugar groups suchas 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; oroligonucleotides having modified bases such as 5-methyl cytosine,2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may beproduced by any method including chemical synthesis or transcription.Once introduced into a cell, the complementary antisense moleculebase-pairs with a naturally occurring nucleic acid sequence produced bythe cell to form duplexes which block either transcription ortranslation. The designation “negative” or “minus” can refer to theantisense strand, and the designation “positive” or “plus” can refer tothe sense strand of a reference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic ENZM, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide” and a “compositioncomprising a given polypeptide” can refer to any composition containingthe given polynucleotide or polypeptide. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotides encoding ENZM or fragments of ENZM may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGEL VIEW fragment assembly system (Accelrys, Burlington Mass.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions. Original Residue Conservative Substitution Ala Gly,Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn,Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Gln Met Leu, Ile Phe His, Met, Leu, Trp, TyrSer Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu,Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions(exons). Since an exon may represent a structural or functional domainof the encoded protein, new proteins may be assembled through the novelreassortment of stable substructures, thus allowing acceleration of theevolution of new protein functions.

A “fragment” is a unique portion of ENZM or a polynucleotide encodingENZM which can be identical in sequence to, but shorter in length than,the parent sequence. A fragment may comprise up to the entire length ofthe defined sequence, minus one nucleotide/amino acid residue. Forexample, a fragment may comprise from about 5 to about 1000 contiguousnucleotides or amino acid residues. A fragment used as a probe, primer,antigen, therapeutic molecule, or for other purposes, may be at least 5,10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule. Forexample, a polypeptide fragment may comprise a certain length ofcontiguous amino acids selected from the first 250 or 500 amino acids(or first 25% or 50%) of a polypeptide as shown in a certain definedsequence. Clearly these lengths are exemplary, and any length that issupported by the specification, including the Sequence Listing, tables,and figures, may be encompassed by the present embodiments.

A fragment of SEQ ID NO:54-106 can comprise a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:54-106,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:54-106 can beemployed in one or more embodiments of methods of the invention, forexample, in hybridization and amplification technologies and inanalogous methods that distinguish SEQ ID NO:54-106 from relatedpolynucleotides. The precise length of a fragment of SEQ ID NO:54-106and the region of SEQ ID NO:54-106 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A fragment of SEQ ID NO: 1-53 is encoded by a fragment of SEQ IDNO:54-106. A fragment of SEQ ID NO:1-53 can comprise a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-53. Forexample, a fragment of SEQ ID NO:1-53 can be used as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-53. The precise length of a fragment of SEQ ID NO:1-53 andthe region of SEQ ID NO:1-53 to which the fragment corresponds can bedetermined based on the intended purpose for the fragment using one ormore analytical methods described herein or otherwise known in the art.

A “full length” polynucleotide is one containing at least a translationinitiation codon (e.g., methionine) followed by an open reading frameand a translation termination codon. A “full length” polynucleotidesequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, alternatively, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of identical residuematches between at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing one or more computer algorithms or programs known in the art ordescribed herein. For example, percent identity can be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program. This program ispart of the LASERGENE software package, a suite of molecular biologicalanalysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described inHiggins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins,D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments ofpolynucleotide sequences, the default parameters are set as follows:Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The“weighted” residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms which can be used is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410),which is available from several sources, including the NCBI, Bethesda,Md., and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. TheBLAST software suite includes various sequence analysis programsincluding “blastn,” that is used to align a known polynucleotidesequence with other polynucleotide sequences from a variety ofdatabases. Also available is a tool called “BLAST 2 Sequences” that isused for direct pairwise comparison of two nucleotide sequences. “BLAST2 Sequences” can be accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of identical residuematches between at least two polypeptide sequences aligned using astandardized algorithm. Methods of polypeptide sequence alignment arewell-known. Some alignment methods take into account conservative aminoacid substitutions. Such conservative substitutions, explained in moredetail above, generally preserve the charge and hydrophobicity at thesite of substitution, thus preserving the structure (and thereforefunction) of the polypeptide. The phrases “percent similarity” and “%similarity,” as applied to polypeptide sequences, refer to thepercentage of residue matches, including identical residue matches andconservative substitutions, between at least two polypeptide sequencesaligned using a standardized algorithm. In contrast, conservativesubstitutions are not included in the calculation of percent identitybetween polypeptide sequences.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. and D. W. Russell (2001;Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold SpringHarbor Press, Cold Spring Harbor N.Y., ch. 9).

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acids by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C₀t or R₀t analysis) or formed between one nucleic acid presentin solution and another nucleic acid immobilized on a solid support(e.g., paper, membranes, filters, chips, pins or glass slides, or anyother appropriate substrate to which cells or their nucleic acids havebeen fixed).

The words “insertion” and “addition” refer to changes in an amino acidor polynucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofENZM which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of ENZMwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, antibodies, or other chemical compoundson a substrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, antibody, or other chemical compound having a unique anddefined position on a microarray.

The term “modulate” refers to a change in the activity of ENZM. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of ENZM.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an ENZM may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of ENZM.

“Probe” refers to nucleic acids encoding ENZM, their complements, orfragments thereof, which are used to detect identical, allelic orrelated nucleic acids. Probes are isolated oligonucleotides orpolynucleotides attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. “Primers” are short nucleic acids, usually DNAoligonucleotides, which may be annealed to a target polynucleotide bycomplementary base-pairing. The primer may then be extended along thetarget DNA strand by a DNA polymerase enzyme. Primer pairs can be usedfor amplification (and identification) of a nucleic acid, e.g., by thepolymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in, forexample, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, ColdSpring Harbor N.Y.), Ausubel, F. M. et al. (1999; Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, New York N.Y.), andInnis, M. et al. (1990; PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif.). PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a nucleic acid that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook and Russell (supra). The term recombinant includes nucleicacids that have been altered solely by addition, substitution, ordeletion of a portion of the nucleic acid. Frequently, a recombinantnucleic acid may include a nucleic acid sequence operably linked to apromoter sequence. Such a recombinant nucleic acid may be part of avector that is used, for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA molecule, is composed of thesame linear sequence of nucleotides as the reference DNA molecule withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining ENZM, nucleic acids encoding ENZM, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably atleast about 75% free, and most preferably at least about 90% free fromother components with which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” or “expression profile” refers to the collectivepattern of gene expression by a particular cell type or tissue undergiven conditions at a given time.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. In another embodiment, the nucleicacid can be introduced by infection with a recombinant viral vector,such as a lentiviral vector (Lois, C. et al. (2002) Science295:868-872). The term genetic manipulation does not include classicalcross-breeding, or in vitro fertilization, but rather is directed to theintroduction of a recombinant DNA molecule. The transgenic organismscontemplated in accordance with the present invention include bacteria,cyanobacteria, fungi, plants and animals. The isolated DNA of thepresent invention can be introduced into the host by methods known inthe art, for example infection, transfection, transformation ortransconjugation. Techniques for transferring the DNA of the presentinvention into such organisms are widely known and provided inreferences such as Sambrook and Russell (supra).

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotides that vary from onespecies to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity or sequencesimilarity to the particular polypeptide sequence over a certain lengthof one of the polypeptide sequences using blastp with the “BLAST 2Sequences” tool Version 2.0.9 (May, 7, 1999) set at default parameters.Such a pair of polypeptides may show, for example, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity or sequence similarity over a certain defined lengthof one of the polypeptides.

The Invention

Various embodiments of the invention include new human enzymes (ENZM),the polynucleotides encoding ENZM, and the use of these compositions forthe diagnosis, treatment, or prevention of autoimmune/inflammatorydisorders, infectious disorders, immune deficiencies, disorders ofmetabolism, reproductive disorders, neurological disorders,cardiovascular disorders, eye disorders, and cell proliferativedisorders, including cancer.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide embodiments of the invention. Each polynucleotide andits corresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown. Column 6 shows theIncyte ID numbers of physical, full length clones corresponding to thepolypeptide and polynucleotide sequences of the invention. The fulllength clones encode polypeptides which have at least 95% sequenceidentity to the polypeptide sequences shown in column 3.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database and the PROTEOME database. Columns 1 and 2 show thepolypeptide sequence identification number (Polypeptide SEQ ID NO:) andthe corresponding Incyte polypeptide sequence number (Incyte PolypeptideID) for polypeptides of the invention. Column 3 shows the GenBankidentification number (GenBank ID NO:) of the nearest GenBank homologand the PROTEOME database identification numbers (PROTEOME ID NO:) ofthe nearest PROTEOME database homologs. Column 4 shows the probabilityscores for the matches between each polypeptide and its homolog(s).Column 5 shows the annotation of the GenBank and PROTEOME databasehomolog(s) along with relevant citations where applicable, all of whichare expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Accelrys, Burlington Mass.).Column 6 shows amino acid residues comprising signature sequences,domains, and motifs. Column 7 shows analytical methods for proteinstructure/function analysis and in some cases, searchable databases towhich the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare enzymes. For example, SEQ ID NO:1 is 100% identical, from residueD155 to residue T409, to human cyclic AMP-specific phosphodiesteraseHSPDE4A1A (GenBank ID g3293241) as determined by the Basic LocalAlignment Search Tool (BLAST). (See Table 2.) The BLAST probabilityscore is 8.4e-135, which indicates the probability of obtaining theobserved polypeptide sequence alignment by chance. SEQ ID NO:1 alsocontains a 3′5′-cyclic nucleotide phosphodiesterase domain as determinedby searching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLAST-PRODOM and BLAST-DOMO analyses providefurther corroborative evidence that SEQ ID NO:1 is a phosphodiesterase.In an alternative example, SEQ ID NO:5 is 96% identical, from residue M1to residue L342, to human paraoxonase (GenBank ID g3694659) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 1.0e-179, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:5 has hydrolase activity, and is a paraoxonase thatcan hydrolyze toxic organophosphates, as determined by BLAST analysisusing the PROTEOME database. SEQ ID NO:2 also contains an arylesterasedomain as determined by searching for statistically significant matchesin the hidden Markov model (HMM)-based PFAM database of conservedprotein family domains. (See Table 3.) Data from BLIMPS and BLASTanalyses provide further corroborative evidence that SEQ ID NO:5 is aserum aromatic hydrolase. In an alternative example, SEQ ID NO:6 is 98%identical, from residue M1 to residue L411, to human2-amino-3-ketobutyrate-CoA ligase (GenBank ID g3342906) as determined bythe Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 3.9e-217, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:6 has transferase activity, and is a 2-amino-3-ketobutyrate CoenzymeA ligase as determined by BLAST analysis using the PROTEOME database.SEQ ID NO:6 also contains an aminotransferase domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS, PROFILESCAN and BLAST analyses providefurther corroborative evidence that SEQ ID NO:6 is a2-amino-3-ketobutyrate Coenzyme A ligase. In an alternative example, SEQID NO:12 is 100% identical, from residue M1 to residue V117 and 99%identical, from residue A115 to residue L254, to human3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (GenBank ID g14714839)as determined by the Basic Local Alignment Search Tool (BLAST). (SeeTable 2.) The BLAST probability score is 3.3e-129, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:12 is localized to mitochondria, has lyase activity,and is a 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase thatfunctions in energy metabolism, ketogenesis and leucine catabolism, asdetermined by BLAST analysis using the PROTEOME database. SEQ ID NO:12also contains an HMGL (hydroxymethylglutaryl-CoA lyase)-like domain asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS, BLAST and MOTIFSanalyses provide further corroborative evidence that SEQ ID NO:12 is ahydroxymethylglutaryl-CoA lyase. In an alternative example, SEQ ID NO:13is 99% identical, from residue M1 to residue Y311 and 94% identical,from residue E303 to residue K374, to human farnesyl diphosphatesynthase (GenBank ID g14603061) as determined by the Basic LocalAlignment Search Tool (BLAST). (See Table 2.) The BLAST probabilityscore is 1.9e-202, which indicates the probability of obtaining theobserved polypeptide sequence alignment by chance. SEQ ID NO:13 hastransferase activity, and is a farnesyl diphosphate synthase thatfunctions in cholesterol biosynthesis, as determined by BLAST analysisusing the PROTEOME database. SEQ ID NO:13 also contains a polyprenylsynthetase domain as determined by searching for statisticallysignificant matches in the hidden Markov model (HMM)-based PFAM databaseof conserved protein family domains. (See Table 3.) Data from BLIMPS andBLAST analyses provide further corroborative evidence that SEQ ID NO:13is a farnesyl pyrophosphate synthetase. In an alternative example, SEQID NO:17 is 92% identical, from residue G19 to residue V338 and is 100%identical from residue M1 to residue Q46, to human very-long-chainacyl-CoA dehydrogenase (GenBank ID g790447) as determined by the BasicLocal Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 1.1e-175, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. Inaddition, as determined by BLAST analysis using the PROTEOME database,SEQ ID NO:17 is localized to the mitochondria, has oxidoreductaseactivity, and is homologous to human very long chain acyl-Coenzyme Adehydrogenase, which oxidizes straight chain acyl-CoAs in the initialstep of fatty acid beta-oxidation, and where deficiencies due to themutation in the gene cause sudden infant death syndrome and hypertrophiccardiomyopathy (PROTEOME ID NO:339036|ACADVL). SEQ ID NO:17 alsocontains acyl-CoA dehydrogenase N-terminal and middle domains asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS, MOTIFS, PROFILESCAN,and additional BLAST analyses provide further corroborative evidencethat SEQ ID NO:4 is an acyl-CoA dehydrogenase. In an alternativeexample, SEQ ID NO:25 is 99% identical, from residue M1 to residue M608,to human phosphoenolpyruvate carboxykinase 2 (GenBank ID g12655193) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 0.0, which indicates the probabilityof obtaining the observed polypeptide sequence alignment by chance. SEQID NO:25 is a phosphoenolpyruvate carboxykinase, as determined by BLASTanalysis using the PROTEOME database. SEQ ID NO:6 also contains aphosphoenolpyruvate carboxykinase domain as determined by searching forstatistically significant matches in the hidden Markov model (HMM)-basedPFAM database of conserved protein family domains. (See Table 3.) Datafrom BLIMPS, MOTIFS, and PROFILESCAN analyses provide furthercorroborative evidence that SEQ ID NO:25 is a phosphoenolpyruvatecarboxykinase. In an alternative example, SEQ ID NO:33 is 100%identical, from residue M1 to residue Q101 and is 83% identical fromresidue F66 to residue K236, to human NAD(P)H:menadione oxidoreductase(GenBank ID g189246) as determined by the Basic Local Alignment SearchTool (BLAST). (See Table 2.) The BLAST probability scores are 3.3e-48and 1.3E-71 respectively, which indicate the probabilities of obtainingthe observed polypeptide sequence alignments by chance. As determined byBLAST analysis using the PROTEOME database, SEQ ID NO:33 is cytoplasmic,has oxidoreductase activity, and is homologous to quinone reductase(NAD(P)H:menadione oxidoreductase), a cytosolic reductase targetingquinones which functions in stress responses. Human deficiency of thequinone reductase gene is associated with increased benzenehematotoxicity, urolithiasis and various cancers (PROTEOME ID:331838|Rn.11234). SEQ ID NO:33 also contains a NAD(P)H dehydrogenase(quinone) domain as determined by searching for statisticallysignificant matches in the hidden Markov model (HMM-based PFAM databaseof conserved protein family domains. (See Table 3.) Data from additionalBLAST analyses provide further corroborative evidence that SEQ ID NO:33is an oxidoreductase. In an alternative example, SEQ ID NO:34 is 77%identical, from residue M1 to residue S598, to Xenopus laevis Nfr1(GenBank ID g2443331) as determined by the Basic Local Alignment SearchTool (BLAST). (See Table 2.) The BLAST probability score is 3.1e-258,which indicates the probability of obtaining the observed polypeptidesequence alignment by chance. SEQ ID NO:34 is an oxidoreductase, asdetermined by BLAST analysis using the PROTEOME database. SEQ ID NO:34also contains a pyridine nucleotide-disulphide oxidoreductase domain asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS and further BLASTanalyses provide corroborative evidence that SEQ ID NO:34 is anoxidoreductase. In an alternative example, SEQ ID NO:48 is 99%identical, from residue M1 to residue R618, to human long chain acyl-CoAdehydrogenase (GenBank ID g1008852) as determined by the Basic LocalAlignment Search Tool (BLAST). (See Table 2.) The BLAST probabilityscore is 0.0, which indicates the probability of obtaining the observedpolypeptide sequence alignment by chance. SEQ ID NO:48 also has homologyto acyl-Coenzyme A proteins with oxidative function, as determined byBLAST analysis using the PROTEOME database. SEQ ID NO:48 also containsacyl-CoA dehydrogenase domains as determined by searching forstatistically significant matches in the hidden Markov model (HMM)-basedPFAM database of conserved protein families/domains. (See Table 3.) Datafrom BLIMPS, MOTIFS, PROFILESCAN and additional BLAST analyses of thePRODOM and DOMO databases provide further corroborative evidence thatSEQ ID NO:48 is an acyl-CoA dehydrogenase enzyme. In an alternativeexample, SEQ ID NO:51 is identical, from residue M1 to residue M478 withhuman long-chain acyl-CoA dehydrogenase (GenBank ID g790447) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 4.2e-253, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:51 also has homology to long-chain acyl-CoAdehydrogenases (339036|ACADVL) as determined by BLAST analysis using thePROTEOME database. SEQ ID NO:51 also contains acyl-CoA dehydrogenasedomains as determined by searching for statistically significant matchesin the hidden Markov model (HMM)-based PFAM database of conservedprotein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, andPROFILESCAN analyses provide further corroborative evidence that SEQ IDNO:51 is a splice variant of acyl-CoA dehydrogenases. SEQ ID NO:2-4, SEQID NO:7-11, SEQ ID NO:14-16, SEQ ID NO:18-24, SEQ ID NO:26-32, SEQ IDNO:35-47, SEQ ID NO:49-50, and SEQ ID NO:52-53 were analyzed andannotated in a similar manner. The algorithms and parameters for theanalysis of SEQ ID NO:1-53 are described in Table 7.

As shown in Table 4, the full length polynucleotide embodiments wereassembled using cDNA sequences or coding (exon) sequences derived fromgenomic DNA, or any combination of these two types of sequences. Column1 lists the polynucleotide sequence identification number(Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotideconsensus sequence number (Incyte ID) for each polynucleotide of theinvention, and the length of each polynucleotide sequence in basepairs.Column 2 shows the nucleotide start (5′) and stop (3′) positions of thecDNA and/or genomic sequences used to assemble the full lengthpolynucleotide embodiments, and of fragments of the polynucleotideswhich are useful, for example, in hybridization or amplificationtechnologies that identify SEQ ID NO:54-106 or that distinguish betweenSEQ ID NO:54-106 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may referspecifically, for example, to Incyte cDNAs derived from tissue-specificcDNA libraries or from pooled cDNA libraries. Alternatively, thepolynucleotide fragments described in column 2 may refer to GenBankcDNAs or ESTs which contributed to the assembly of the full lengthpolynucleotides. In addition, the polynucleotide fragments described incolumn 2 may identify sequences derived from the ENSEMBL (The SangerCentre, Cambridge, UK) database (i.e., those sequences including thedesignation “ENST”). Alternatively, the polynucleotide fragmentsdescribed in column 2 may be derived from the NCBI RefSeq NucleotideSequence Records Database (i.e., those sequences including thedesignation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records(i.e., those sequences including the designation “NP”). Alternatively,the polynucleotide fragments described in column 2 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm For example, a polynucleotide sequenceidentified as FL_XXXXXX_N_(1—)N_(2—)YYYYY_ N_(3—)N₄ represents a“stitched” sequence in which XXXXXX is the identification number of thecluster of sequences to which the algorithm was applied, and YYYYY isthe number of the prediction generated by the algorithm, and N_(1,2,3) .. . , if present, represent specific exons that may have been manuallyedited during analysis (See Example V). Alternatively, thepolynucleotide fragments in column 2 may refer to assemblages of exonsbrought together by an “exon-stretching” algorithm. For example, apolynucleotide sequence identified as FLXXXXXX_gAAAAAA_gBBBBB_(—)1_N isa “stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may beused in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V). Prefix Type of analysis and/or examples of programs GNN,GFG, Exon prediction from genomic sequences using, for ENST example,GENSCAN (Stanford University, CA, USA) or FGENES (Computer GenomicsGroup, The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis ofgenomic sequences. FL Stitched or stretched genomic sequences (seeExample V). INCY Full length transcript and exon prediction from mappingof EST sequences to the genome. Genomic location and EST compositiondata are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverageshown in Table 4 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotides which were assembled using Incyte cDNA sequences. Therepresentative cDNA library is the Incyte cDNA library which is mostfrequently represented by the Incyte cDNA sequences which were used toassemble and confirm the above polynucleotides. The tissues and vectorswhich were used to construct the cDNA libraries shown in Table 5 aredescribed in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found inpolynucleotide sequences of the invention, along with allele frequenciesin different human populations. Columns 1 and 2 show the polynucleotidesequence identification number (SEQ ID NO:) and the corresponding Incyteproject identification number (PID) for polynucleotides of theinvention. Column 3 shows the Incyte identification number for the ESTin which the SNP was detected (EST ID), and column 4 shows theidentification number for the SNP (SNP ED). Column 5 shows the positionwithin the EST sequence at which the SNP is located (EST SNP), andcolumn 6 shows the position of the SNP within the full-lengthpolynucleotide sequence (CB1 SNP). Column 7 shows the allele found inthe EST sequence. Columns 8 and 9 show the two alleles found at the SNPsite. Column 10 shows the amino acid encoded by the codon including theSNP site, based upon the allele found in the EST. Columns 11-14 show thefrequency of allele 1 in four different human populations. An entry ofn/d (not detected) indicates that the frequency of allele 1 in thepopulation was too low to be detected, while n/a (not available)indicates that the allele frequency was not determined for thepopulation.

The invention also encompasses ENZM variants. Various embodiments ofENZM variants can have at least about 80%, at least about 90%, or atleast about 95% amino acid sequence identity to the ENZM amino acidsequence, and can contain at least one functional or structuralcharacteristic of ENZM.

Various embodiments also encompass polynucleotides which encode ENZM. Ina particular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:54-106, which encodes ENZM. The polynucleotide sequences of SEQ IDNO:54-106, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses variants of a polynucleotide encodingENZM. In particular, such a variant polynucleotide will have at leastabout 70%, or alternatively at least about 85%, or even at least about95% polynucleotide sequence identity to a polynucleotide encoding ENZM.A particular aspect of the invention encompasses a variant of apolynucleotide comprising a sequence selected from the group consistingof SEQ ID NO:54-106 which has at least about 70%, or alternatively atleast about 85%, or even at least about 95% polynucleotide sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO:54-106. Any one of the polynucleotide variants describedabove can encode a polypeptide which contains at least one functional orstructural characteristic of ENZM.

In addition, or in the alternative, a polynucleotide variant of theinvention is a splice variant of a polynucleotide encoding ENZM. Asplice variant may have portions which have significant sequenceidentity to a polynucleotide encoding ENZM, but will generally have agreater or lesser number of polynucleotides due to additions ordeletions of blocks of sequence arising from alternate splicing of exonsduring mRNA processing. A splice variant may have less than about 70%,or alternatively less than about 60%, or alternatively less than about50% polynucleotide sequence identity to a polynucleotide encoding ENZMover its entire length; however, portions of the splice variant willhave at least about 70%, or alternatively at least about 85%, oralternatively at least about 95%, or alternatively 100% polynucleotidesequence identity to portions of the polynucleotide encoding ENZM. Forexample, a polynucleotide comprising a sequence of SEQ ID NO:93 and apolynucleotide comprising a sequence of SEQ ID NO:54 are splice variantsof each other; a polynucleotide comprising a sequence of SEQ ID NO:99and a polynucleotide comprising a sequence of SEQ ID NO:59 are splicevariants of each other; a polynucleotide comprising a sequence of SEQ IDNO:98 and a polynucleotide comprising a sequence of SEQ ID NO:62 aresplice variants of each other; a polynucleotide comprising a sequence ofSEQ ID NO:102 and a polynucleotide comprising a sequence of SEQ ID NO:66are splice variants of each other; a polynucleotide comprising asequence of SEQ ID NO:100, a polynucleotide comprising a sequence of SEQID NO:101, a polynucleotide comprising a sequence of SEQ ID NO:104, anda polynucleotide comprising a sequence of SEQ ID NO:70 are splicevariants of each other; a polynucleotide comprising a sequence of SEQ IDNO:94, a polynucleotide comprising a sequence of SEQ ID NO:95, apolynucleotide comprising a sequence of SEQ ID NO:96, and apolynucleotide comprising a sequence of SEQ ID NO:73 are splice variantsof each other; a polynucleotide comprising a sequence of SEQ ID NO:97and a polynucleotide comprising a sequence of SEQ ID NO:75 are splicevariants of each other; a polynucleotide comprising a sequence of SEQ IDNO:105 and a polynucleotide comprising a sequence of SEQ ID NO:79 aresplice variants of each other; a polynucleotide comprising a sequence ofSEQ ID NO:103, a polynucleotide comprising a sequence of SEQ ID NO:106,and a polynucleotide comprising a sequence of SEQ ID NO:89 are splicevariants of each other; a polynucleotide comprising a sequence of SEQ IDNO:57 and a polynucleotide comprising a sequence of SEQ ID NO:58 aresplice variants of each other; a polynucleotide comprising a sequence ofSEQ ID NO:67, a polynucleotide comprising a sequence of SEQ ID NO:68, apolynucleotide comprising a sequence of SEQ ID NO:71, and apolynucleotide comprising a sequence of SEQ ID NO:72 are splice variantsof each other; and a polynucleotide comprising a sequence of SEQ IDNO:82, and a polynucleotide comprising a sequence of SEQ ID NO:83 aresplice variants of each other. Any one of the splice variants describedabove can encode a polypeptide which contains at least one functional orstructural characteristic of ENZM.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding ENZM, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring ENZM, and all suchvariations are to be considered as being specifically disclosed.

Although polynucleotides which encode ENZM and its variants aregenerally capable of hybridizing to polynucleotides encoding naturallyoccurring ENZM under appropriately selected conditions of stringency, itmay be advantageous to produce polynucleotides encoding ENZM or itsderivatives possessing a substantially different codon usage, e.g.,inclusion of non-naturally occurring codons. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encoding ENZMand its derivatives without altering the encoded amino acid sequencesinclude the production of RNA transcripts having more desirableproperties, such as a greater half-life, than transcripts produced fromthe naturally occurring sequence.

The invention also encompasses production of polynucleotides whichencode ENZM and ENZM derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic polynucleotide maybe inserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a polynucleotideencoding ENZM or any fragment thereof.

Embodiments of the invention can also include polynucleotides that arecapable of hybridizing to the claimed polynucleotides, and, inparticular, to those having the sequences shown in SEQ ID NO:54-106 andfragments thereof, under various conditions of stringency (Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511). Hybridization conditions,including annealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Biosciences, Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Invitrogen, CarlsbadCalif.). Preferably, sequence preparation is automated with machinessuch as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.),PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST800 thermal cycler (Applied Biosystems). Sequencing is then carried outusing either the ABI 373 or 377 DNA sequencing system (AppliedBiosystems), the MEGABACE 1000 DNA sequencing system (AmershamBiosciences), or other systems known in the art. The resulting sequencesare analyzed using a variety of algorithms which are well known in theart (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) MolecularBiology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).

The nucleic acids encoding ENZM may be extended utilizing a partialnucleotide sequence and employing various PCR-based methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector (Sarkar, G.(1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, usesprimers that extend in divergent directions to amplify unknown sequencefrom a circularized template. The template is derived from restrictionfragments comprising a known genomic locus and surrounding sequences(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method,capture PCR, involves PCR amplification of DNA fragments adjacent toknown sequences in human and yeast artificial chromosome DNA(Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In thismethod, multiple restriction enzyme digestions and ligations may be usedto insert an engineered double-stranded sequence into a region ofunknown sequence before performing PCR. Other methods which may be usedto retrieve unknown sequences are known in the art (Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotides or fragmentsthereof which encode ENZM may be cloned in recombinant DNA moleculesthat direct expression of ENZM, or fragments or functional equivalentsthereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other polynucleotides which encode substantially thesame or a functionally equivalent polypeptides may be produced and usedto express ENZM.

The polynucleotides of the invention can be engineered using methodsgenerally known in the art in order to alter ENZM-encoding sequences fora variety of purposes including, but not limited to, modification of thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, oligonucleotide-mediated site-directedmutagenesis may be used to introduce mutations that create newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of ENZM, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding ENZM may be synthesized,in whole or in part, using one or more chemical methods well known inthe art (Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser.7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).Alternatively, ENZM itself or a fragment thereof may be synthesizedusing chemical methods known in the art. For example, peptide synthesiscan be performed using various solution-phase or solid-phase techniques(Creighton, T. (1984) Proteins, Structures and Molecular Properties, WHFreeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science269:202-204). Automated synthesis may be achieved using the ABI 431Apeptide synthesizer (Applied Biosystems). Additionally, the amino acidsequence of ENZM, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990)Methods Enzymol. 182:392-421). The composition of the synthetic peptidesmay be confirmed by amino acid analysis or by sequencing (Creighton,supra, pp. 28-53).

In order to express a biologically active ENZM, the polynucleotidesencoding ENZM or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesencoding ENZM. Such elements may vary in their strength and specificity.Specific initiation signals may also be used to achieve more efficienttranslation of polynucleotides encoding ENZM. Such signals include theATG initiation codon and adjacent sequences, e.g. the Kozak sequence. Incases where a polynucleotide sequence encoding ENZM and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used (Scharf,D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing polynucleotides encoding ENZMand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (Sambrook and Russell,supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).

A variety of expression vector/host systems may be utilized to containand express polynucleotides encoding ENZM. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrookand Russell, supra; Ausubel et al., supra; Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet.15:345-355). Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of polynucleotides to the targeted organ, tissue,or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther.5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344;Buller, R. M. et al. (1985) Nature 317:813-815; McGregor, D. P. et al.(1994) Mol. Immunol. 31:219-226; Verma, I. M. and N. Somia (1997) Nature389:239-242). The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotides encodingENZM. For example, routine cloning, subcloning, and propagation ofpolynucleotides encoding ENZM can be achieved using a multifunctional E.coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1plasmid (Invitrogen). Ligation of polynucleotides encoding ENZM into thevector's multiple cloning site disrupts the lacZ gene, allowing acolorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509). When large quantities of ENZM are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofENZM may be used. For example, vectors containing the strong, inducibleSP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of ENZM. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign polynucleotide sequences intothe host genome for stable propagation (Ausubel et al., supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al.(1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of ENZM. Transcription ofpolynucleotides encoding ENZM may be driven by viral promoters, e.g.,the 35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J.et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructscan be introduced into plant cells by direct DNA transformation orpathogen-mediated transfection (The McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,polynucleotides encoding ENZM may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses ENZM in host cells (Logan, J. and T. Shenk (1984) Proc. Natl.Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increase.expression in mammalian host cells. SV40 or EBV-based vectors may alsobe used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997)Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems,stable expression of ENZM in cell lines is preferred. For example,polynucleotides encoding ENZM can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ and apr⁻ cells, respectively (Wigler, M. et al. (1977) Cell11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Wigler, M. et al.(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. etal. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, greenfluorescent proteins (GFP; Clontech), β-glucuronidase and its substrateβ-glucuronide, or luciferase and its substrate luciferin may be used.These markers can be used not only to identify transformants, but alsoto quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes, C. A. (1995) MethodsMol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingENZM is inserted within a marker gene sequence, transformed cellscontaining polynucleotides encoding ENZM can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding ENZM under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding ENZM andthat express ENZM may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of ENZMusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on ENZMis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art (Hampton, R. et al. (1990)Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn.,Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding ENZM includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, polynucleotides encodingENZM, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Biosciences, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with polynucleotides encoding ENZM may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeENZM may be designed to contain signal sequences which direct secretionof ENZM through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted polynucleotides or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding ENZM may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric ENZMprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of ENZM activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the ENZM encodingsequence and the heterologous protein sequence, so that ENZM may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel et al. (supra, ch. 10 and 16). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

In another embodiment, synthesis of radiolabeled ENZM may be achieved invitro using the TNT rabbit reticulocyte lysate or wheat germ extractsystem (Promega). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, for example, ³⁵S-methionine.

ENZM, fragments of ENZM, or variants of ENZM may be used to screen forcompounds that specifically bind to ENZM. One or more test compounds maybe screened for specific binding to ENZM. In various embodiments, 1, 2,3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened forspecific binding to ENZM. Examples of test compounds can includeantibodies, anticalins, oligonucleotides, proteins (e.g., ligands orreceptors), or small molecules.

In related embodiments, variants of ENZM can be used to screen forbinding of test compounds, such as antibodies, to ENZM, a variant ofENZM, or a combination of ENZM and/or one or more variants ENZM. In anembodiment, a variant of ENZM can be used to screen for compounds thatbind to a variant of ENZM, but not to ENZM having the exact sequence ofa sequence of SEQ ID NO:1-53. ENZM variants used to perform suchscreening can have a range of about 50% to about 99% sequence identityto ENZM, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%,and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific bindingto ENZM can be closely related to the natural ligand of ENZM, e.g., aligand or fragment thereof, a natural substrate, a structural orfunctional mimetic, or a natural binding partner (Coligan, J. E. et al.(1991) Current Protocols in Immunology 1(2):Chapter 5). In anotherembodiment, the compound thus identified can be a natural ligand of areceptor ENZM (Howard, A. D. et al. (2001) Trends Pharmacol. Sci. 22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specificbinding to ENZM can be closely related to the natural receptor to whichENZM binds, at least a fragment of the receptor, or a fragment of thereceptor including all or a portion of the ligand binding site orbinding pocket. For example, the compound may be a receptor for ENZMwhich is capable of propagating a signal, or a decoy receptor for ENZMwhich is not capable. of propagating a signal (Ashkenazi, A. and V. M.Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al.(2001) Trends Immunol. 22:328-336). The compound can be rationallydesigned using known techniques. Examples of such techniques includethose used to construct the compound etanercept (ENBREL; Amgen Inc.,Thousand Oaks Calif.), which is efficacious for treating rheumatoidarthritis in humans. Etanercept is an engineered p75 tumor necrosisfactor (TNF) receptor dimer linked to the Fc portion of human IgG₁(Taylor, P. C. et at. (2001) Curr. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar or,alternatively, different specificities can be screened for specificbinding to ENZM, fragments of ENZM, or variants of ENZM. The bindingspecificity of the antibodies thus screened can thereby be selected toidentify particular fragments or variants of ENZM. In one embodiment, anantibody can be selected such that its binding specificity allows forpreferential identification of specific fragments or variants of ENZM.In another embodiment, an antibody can be selected such that its bindingspecificity allows for preferential diagnosis of a specific disease orcondition having increased, decreased, or otherwise abnormal productionof ENZM.

In an embodiment, anticalins can be screened for specific binding toENZM, fragments of ENZM, or variants of ENZM. Anticalins areligand-binding proteins that have been constructed based on a lipocalinscaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184;Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architectureof lipocalins can include a beta-barrel having eight antiparallelbeta-strands, which supports four loops at its open end. These loopsform the natural ligand-binding site of the lipocalins, a site which canbe re-engineered in vitro by amino acid substitutions to impart novelbinding specificities. The amino acid substitutions can be made usingmethods known in the art or described herein, and can includeconservative substitutions (e.g., substitutions that do not alterbinding specificity) or substitutions that modestly, moderately, orsignificantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to,stimulate, or inhibit ENZM involves producing appropriate cells whichexpress ENZM, either as a secreted protein or on the cell membrane.Preferred cells can include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing ENZM or cell membrane fractions which containENZM are then contacted with a test compound and binding, stimulation,or inhibition of activity of either ENZM or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with ENZM,either in solution or affixed to a solid support, and detecting thebinding of ENZM to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to itsnatural ligand and/or to inhibit the binding of its natural ligand toits natural receptors. Examples of such assays include radio-labelingassays such as those described in U.S. Pat. No. 5,914,236 and U.S. Pat.No. 6,372,724. In a related embodiment, one or more amino acidsubstitutions can be introduced into a polypeptide compound (such as areceptor) to improve or alter its ability to bind to its natural ligands(Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). Inanother related embodiment, one or more amino acid substitutions can beintroduced into a polypeptide compound (such as a ligand) to improve oralter its ability to bind to its natural receptors (Cunningham, B. C.and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman,H. B. et al. (1991) J. Biol. Chem. 266:10982-10988).

ENZM, fragments of ENZM, or variants of ENZM may be used to screen forcompounds that modulate the activity of ENZM. Such compounds may includeagonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for ENZMactivity, wherein ENZM is combined with at least one test compound, andthe activity of ENZM in the presence of a test compound is compared withthe activity of ENZM in the absence of the test compound. A change inthe activity of ENZM in the presence of the test compound is indicativeof a compound that modulates the activity of ENZM. Alternatively, a testcompound is combined with an in vitro or cell-free system comprisingENZM under conditions suitable for ENZM activity, and the assay isperformed. In either of these assays, a test compound which modulatesthe activity of ENZM may do so indirectly and need not come in directcontact with the test compound. At least one and up to a plurality oftest compounds may be screened.

In another embodiment, polynucleotides encoding ENZM or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding ENZM may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding ENZM can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding ENZM is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress ENZM, e.g.,by secreting ENZM in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of ENZM and enzymes. In addition,examples of tissues expressing ENZM can be found in Table 6 and can alsobe found in Example XI. Therefore, ENZM appears to play a role inautoimmune/inflammatory disorders, infectious disorders, immunedeficiencies, disorders of metabolism, reproductive disorders,neurological disorders, cardiovascular disorders, eye disorders, andcell proliferative disorders, including cancer. In the treatment ofdisorders associated with increased ENZM expression or activity, it isdesirable to decrease the expression or activity of ENZM. In thetreatment of disorders associated with decreased ENZM expression oractivity, it is desirable to increase the expression or activity ofENZM.

Therefore, in one embodiment, ENZM or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of ENZM. Examples ofsuch disorders include, but are not limited to, anautoimmune/inflammatory disorder such as acquired immunodeficiencysyndrome (AIDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, and trauma; aninfectious disorder such as a viral infection, e.g., caused by anadenovirus (acute respiratory disease, pneumonia), an arenavirus(lymphocytic choriomeningitis), a bunyavirus (Hantavirus), a coronavirus(pneumonia, chronic bronchitis), a hepadnavirus (hepatitis), aherpesvirus (herpes simplex virus, varicella-zoster virus, Epstein-Barrvirus, cytomegalovirus), a flavivirus (yellow fever), an orthomyxovirus(influenza), a papillomavirus (cancer), a paramyxovirus (measles,mumps), a picornovirus (rhinovirus, poliovirus, coxsackie-virus), apolyomavirus (BK virus, JC virus), a poxvirus (smallpox), a reovirus(Colorado tick fever), a retrovirus (human immunodeficiency virus, humanT lymphotropic virus), a rhabdovirus (rabies), a rotavirus(gastroenteritis), and a togavirus (encephalitis, rubella), and abacterial infection, a fungal infection, a parasitic infection, aprotozoal infection, and a helminthic infection; an immune deficiency,such as acquired immunodeficiency syndrome (AIDS), X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; a disorder of metabolism such as Addison's disease,cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarinresistance, cystic fibrosis, diabetes, fatty hepatocirrhosis,fructose-1,6-diphosphatase deficiency, galactosemia, goiter,glucagonoma, glycogen storage diseases, hereditary fructose intolerance,hyperadrenalism, hypoadrenalism, hyperparathyroidism,hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,hypothyroidism, hyperlipidemia, hyperlipemia, a lipid myopathy, alipodystrophy, a lysosomal storage disease, mannosidosis, neuraminidasedeficiency, obesity, pentosuria phenylketonuria, pseudovitaminD-deficiency rickets; a reproductive disorder such as a disorder ofprolactin production, infertility, including tubal disease, ovulatorydefects, and endometriosis, a disruption of the estrous cycle, adisruption of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, uterinefibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis,cancer of the breast, fibrocystic breast disease, and galactorrhea,disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, Peyronie's disease, impotence, carcinoma of the malebreast, and gynecomastia; a neurological disorder such as epilepsy,ischemic cerebrovascular disease, stroke, cerebral neoplasms,Alzheimer's disease, Pick's disease, Huntington's disease, dementia,Parkinson's disease and other extrapyramidal disorders, amyotrophiclateral sclerosis and other motor neuron disorders, progressive neuralmuscular atrophy, retinitis pigmentosa, hereditary ataxias, multiplesclerosis and other demyelinating diseases, bacterial and viralmeningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease; prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome;fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;seasonal affective disorder (SAD); akathesia, amnesia, catatonia,diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, and Tourette's disorder; a cardiovasculardisorder, such as arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery, congestive heartfailure, ischemic heart disease, angina pectoris, myocardial infarction,hypertensive heart disease, degenerative valvular heart disease,calcific aortic valve stenosis, congenitally bicuspid aortic valve,mitral annular calcification, mitral valve prolapse, rheumatic fever andrheumatic heart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, and complications of cardiactransplantation, congenital lung anomalies, atelectasis, pulmonarycongestion and edema, pulmonary embolism, pulmonary hemorrhage,pulmonary infarction, pulmonary hypertension, vascular sclerosis,obstructive pulmonary disease, restrictive pulmonary disease, chronicobstructive pulmonary disease, emphysema, chronic bronchitis, bronchialasthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmalpneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitialdiseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis,desquamative interstitial pneumonitis, hypersensitivity pneumonitis,pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia,diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes,idiopathic pulmonary hemosiderosis, pulmonary involvement incollagen-vascular disorders, pulmonary alveolar proteinosis, lungtumors, inflammatory and noninflammatory pleural effusions,pneumothorax, pleural tumors, drug-induced lung disease,radiation-induced lung disease, and complications of lungtransplantation; an eye disorder such as ocular hypertension andglaucoma; a disorder of cell proliferation such as actinic keratosis,arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixedconnective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnalhemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia;and a cancer, including adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus.

In another embodiment, a vector capable of expressing ENZM or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofENZM including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified ENZM in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of ENZM including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofENZM may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of ENZM including, butnot limited to, those listed above.

In a further embodiment, an antagonist of ENZM may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of ENZM. Examples of such disorders include, butare not limited to, those autoimmune/inflammatory disorders, infectiousdisorders, immune deficiencies, disorders of metabolism, reproductivedisorders, neurological disorders, cardiovascular disorders, eyedisorders, and cell proliferative disorders, including cancer describedabove. In one aspect, an antibody which specifically binds ENZM may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissues whichexpress ENZM.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding ENZM may be administered to a subject to treator prevent a disorder associated with increased expression or activityof ENZM including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody,complementary sequence, or vector embodiments may be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy may be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of ENZM may be produced using methods which are generallyknown in the art. In particular, purified ENZM may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind ENZM. Antibodies to ENZM may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. In an embodiment, neutralizing antibodies (i.e.,those which inhibit dimer formation) can be used therapeutically. Singlechain antibodies (e.g., from camels or llamas) may be potent enzymeinhibitors and may have application in the design of peptide mimetics,and in the development of immuno-adsorbents and biosensors (Muyldermans,S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with ENZM or with any fragment or oligopeptidethereof which has immunogenic properties. Depending on the host species,various adjuvants may be used to increase immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to ENZM have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are substantially identical to a portion of theamino acid sequence of the natural protein. Short stretches of ENZMamino acids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to ENZM may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:3142; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. CellBiol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceENZM-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837;Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for ENZM mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse, W. D. etal. (1989) Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between ENZM and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering ENZM epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for ENZM. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of ENZM-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple ENZM epitopes, represents the average affinity,or avidity, of the antibodies for ENZM. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular ENZM epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theENZM-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of ENZM, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/M1, is generally employed in proceduresrequiring precipitation of ENZM-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable (Catty, supra; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding ENZM,or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding ENZM. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding ENZM (Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press, Totawa N.J.).

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein (Slater, J. E. et al. (1998) J. Allergy Clin.Immunol. 102:469475; Scanlon, K. J. et al. (1995) 9:1288-1296).Antisense sequences can also be introduced intracellularly through theuse of viral vectors, such as retrovirus and adeno-associated virusvectors (Miller, A. D. (1990) Blood 76:271; Ausubel et al., supra;Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Othergene delivery mechanisms include liposome-derived systems, artificialviral envelopes, and other systems known in the art (Rossi, J. J. (1995)Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res.25:2730-2736).

In another embodiment of the invention, polynucleotides encoding ENZMmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV,HCV); fungal parasites, such as Candida albicans and Paracoccidioidesbrasiliensis; and protozoan parasites such as Plasmodium falciparum andTrypanosoma cruzi). In the case where a genetic deficiency in ENZMexpression or regulation causes disease, the expression of ENZM from anappropriate population of transduced cells may alleviate the clinicalmanifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in ENZM are treated by constructing mammalian expressionvectors encoding ENZM and introducing these vectors by mechanical meansinto ENZM-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J.-L. and H. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of ENZMinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT,PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF,PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). ENZMmay be expressed using (i) a constitutively active promoter, (e.g., fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidinekinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding ENZM from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to ENZM expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding ENZM under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding ENZM to cells which have one ormore genetic abnormalities with respect to the expression of ENZM. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999; Annu. Rev. Nutr. 19:511-544) and Verma, I. M. and N. Somia (1997;Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding ENZM to target cells which haveone or more genetic abnormalities with respect to the expression ofENZM. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing ENZM to cells of the central nervoussystem, for which HSV has a tropism. The construction and packaging ofherpes-based vectors are well known to those with ordinary skill in theart. A replication-competent herpes simplex virus (HSV) type 1-basedvector has been used to deliver a reporter gene to the eyes of primates(Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of aHSV-1 virus vector has also been disclosed in detail in U.S. Pat. No.5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”),which is hereby incorporated by reference. U.S. Pat. No. 5,804,413teaches the use of recombinant HSV d92 which consists of a genomecontaining at least one exogenous gene to be transferred to a cell underthe control of the appropriate promoter for purposes including humangene therapy. Also taught by this patent are the construction and use ofrecombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSVvectors, see also Goins, W. F. et al. (1999; J. Virol. 73:519-532) andXu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of clonedherpesvirus sequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding ENZM to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for ENZM into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of ENZM-coding RNAs and the synthesis of high levels ofENZM in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:74-83). The widehost range of alphaviruses will allow the introduction of ENZM into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177).A complementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of RNA moleculesencoding ENZM.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be preparedby any method known in the art for the synthesis of nucleic acidmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA molecules encoding ENZM. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize complementary RNA, constitutively or inducibly, can beintroduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or moreselected polynucleotides of the present invention can be altered,inhibited, decreased, or silenced using RNA interference (RNAi) orpost-transcriptional gene silencing (PTGS) methods known in the art.RNAi is a post-transcriptional mode of gene silencing in whichdouble-stranded RNA (dsRNA) introduced into a targeted cell specificallysuppresses the expression of the homologous gene (i.e., the gene bearingthe sequence complementary to the dsRNA). This effectively knocks out orsubstantially reduces the expression of the targeted gene. PTGS can alsobe accomplished by use of DNA or DNA fragments as well. RNAi methods aredescribed by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T.(2000; Nature 404:804-808). PTGS can also be initiated by introductionof a complementary segment of DNA into the selected tissue using genedelivery and/or viral vector delivery methods described herein or knownin the art.

RNAi can be induced in mammalian cells by the use of small interferingRNA also known as siRNA. SiRNA are shorter segments of dsRNA (typicallyabout 21 to 23 nucleotides in length) that result in vivo from cleavageof introduced dsRNA by the action of an endogenous ribonuclease. SiRNAappear to be the mediators of the RNAi effect in mammals. The mosteffective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3′overhangs. The use of siRNA for inducing RNAi in mammalian cells isdescribed by Elbashir, S. M. et al. (2001; Nature 411:494-498).

SiRNA can either be generated indirectly by introduction of dsRNA intothe targeted cell, or directly by mammalian transfection methods andagents described herein or known in the art (such as liposome-mediatedtransfection, viral vector methods, or other polynucleotidedelivery/introductory methods). Suitable SiRNAs can be selected byexamining a transcript of the target polynucleotide (e.g., mRNA) fornucleotide sequences downstream from the AUG start codon and recordingthe occurrence of each nucleotide and the 3′ adjacent 19 to 23nucleotides as potential siRNA target sites, with sequences having a 21nucleotide length being preferred. Regions to be avoided for targetsiRNA sites include the 5′ and 3′ untranslated regions (UTRs) andregions near the start codon (within 75 bases), as these may be richerin regulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNAendonuclease complex. The selected target sites for siRNA can then becompared to the appropriate genome database (e.g., human, etc.) usingBLAST or other sequence comparison algorithms known in the art. Targetsequences with significant homology to other coding sequences can beeliminated from consideration. The selected SiRNAs can be produced bychemical synthesis methods known in the art or by in vitro transcriptionusing commercially available methods and kits such as the SILENCER siRNAconstruction kit (Ambion, Austin Tex.).

In alternative embodiments, long-term gene silencing and/or RNAi effectscan be induced in selected tissue using expression vectors thatcontinuously express siRNA. This can be accomplished using expressionvectors that are engineered to express hairpin RNAs (shRNAs) usingmethods known in the art (see, e.g., Brummelkamp, T. R. et al. (2002)Science 296:550-553; and Paddison, P. J. et al. (2002) Genes Dev.16:948-958). In these and related embodiments, shRNAs can be deliveredto target cells using expression vectors known in the art. An example ofa suitable expression vector for delivery of siRNA is thePSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to thetarget tissue, shRNAs are processed in vivo into siRNA-like moleculescapable of carrying out gene-specific silencing.

In various embodiments, the expression levels of genes targeted by RNAior PTGS methods can be determined by assays for mRNA and/or proteinanalysis. Expression levels of the mRNA of a targeted gene, can bedetermined by northern analysis methods using, for example, theNORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; byreal time PCR methods; and by other RNA/polynucleotide assays known inthe art or described herein. Expression levels of the protein encoded bythe targeted gene can be determined by Western analysis using standardtechniques known in the art.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding ENZM. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased ENZMexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding ENZM may be therapeuticallyuseful, and in the treatment of disorders associated with decreased ENZMexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding ENZM may be therapeuticallyuseful.

In various embodiments, one or more test compounds may be screened foreffectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding ENZM isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding ENZM are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingENZM. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol.15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of ENZM,antibodies to ENZM, and mimetics, agonists, antagonists, or inhibitorsof ENZM.

In various embodiments, the compositions described herein, such aspharmaceutical compositions, may be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal,enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery allows administration without needleinjection, and obviates the need for potentially toxic penetrationenhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising ENZM or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, ENZM or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example ENZM or fragments thereof, antibodies of ENZM,and agonists, antagonists or inhibitors of ENZM, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅/ED₅₀ ratio. Compositions which exhibit large therapeutic indicesare preferred. The data obtained from cell culture assays and animalstudies are used to formulate a range of dosage for human use. Thedosage contained in such compositions is preferably within a range ofcirculating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind ENZM may beused for the diagnosis of disorders characterized by expression of ENZM,or in assays to monitor patients being treated with ENZM or agonists,antagonists, or inhibitors of ENZM. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for ENZM include methods which utilizethe antibody and a label to detect ENZM in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring ENZM, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of ENZM expression. Normal or standard values for ENZMexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to ENZM under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of ENZM expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding ENZMmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotides, complementary RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantify geneexpression in biopsied tissues in which expression of ENZM may becorrelated with disease. The diagnostic assay may be used to determineabsence, presence, and excess expression of ENZM, and to monitorregulation of ENZM levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotides, including genomic sequences, encoding ENZM orclosely related molecules may be used to identify nucleic acid sequenceswhich encode ENZM. The specificity of the probe, whether it is made froma highly specific region, e.g., the 5′ regulatory region, or from a lessspecific region, e.g., a conserved motif, and the stringency of thehybridization or amplification will determine whether the probeidentifies only naturally occurring sequences encoding ENZM, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the ENZM encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:54-106 or fromgenomic sequences including promoters, enhancers, and introns of theENZM gene.

Means for producing specific hybridization probes for polynucleotidesencoding ENZM include the cloning of polynucleotides encoding ENZM orENZM derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotides encoding ENZM may be used for the diagnosis of disordersassociated with expression of ENZM. Examples of such disorders include,but are not limited to, an autoimmune/inflammatory disorder such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, and trauma; aninfectious disorder such as a viral infection, e.g., caused by anadenovirus (acute respiratory disease, pneumonia), an arenavirus(lymphocytic choriomeningitis), a bunyavirus (Hantavirus), a coronavirus(pneumonia, chronic bronchitis), a hepadnavirus (hepatitis), aherpesvirus (herpes simplex virus, varicella-zoster virus, Epstein-Barrvirus, cytomegalovirus), a flavivirus (yellow fever), an orthomyxovirus(influenza), a papillomavirus (cancer), a paramyxovirus (measles,mumps), a picornovirus (rhinovirus, poliovirus, coxsackie-virus), apolyomavirus (BK virus, JC virus), a poxvirus (smallpox), a reovirus(Colorado tick fever), a retrovirus (human immunodeficiency virus, humanT lymphotropic virus), a rhabdovirus (rabies), a rotavirus(gastroenteritis), and a togavirus (encephalitis, rubella), and abacterial infection, a fungal infection, a parasitic infection, aprotozoal infection, and a helminthic infection; an immune deficiency,such as acquired immunodeficiency syndrome (AIDS), X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; a disorder of metabolism such as Addison's disease,cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarinresistance, cystic fibrosis, diabetes, fatty hepatocirrhosis,fructose-1,6-diphosphatase deficiency, galactosemia, goiter,glucagonoma, glycogen storage diseases, hereditary fructose intolerance,hyperadrenalism, hypoadrenalism, hyperparathyroidism,hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,hypothyroidism, hyperlipidemia, hyperlipemia, a lipid myopathy, alipodystrophy, a lysosomal storage disease, mannosidosis, neuraminidasedeficiency, obesity, pentosuria phenylketonuria, pseudovitaminD-deficiency rickets; a reproductive disorder such as a disorder ofprolactin production, infertility, including tubal disease, ovulatorydefects, and endometriosis, a disruption of the estrous cycle, adisruption of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, uterinefibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis,cancer of the breast, fibrocystic breast disease, and galactorrhea,disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, Peyronie's disease, impotence, carcinoma of the malebreast, and gynecomastia; a neurological disorder such as epilepsy,ischemic cerebrovascular disease, stroke, cerebral neoplasms,Alzheimer's disease, Pick's disease, Huntington's disease, dementia,Parkinson's disease and other extrapyramidal disorders, amyotrophiclateral sclerosis and other motor neuron disorders, progressive neuralmuscular atrophy, retinitis pigmentosa, hereditary ataxias, multiplesclerosis and other demyelinating diseases, bacterial and viralmeningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease; prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome;fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;seasonal affective disorder (SAD); akathesia, amnesia, catatonia,diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, and Tourette's disorder; a cardiovasculardisorder, such as arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery, congestive heartfailure, ischemic heart disease, angina pectoris, myocardial infarction,hypertensive heart disease, degenerative valvular heart disease,calcific aortic valve stenosis, congenitally bicuspid aortic valve,mitral annular calcification, mitral valve prolapse, rheumatic fever andrheumatic heart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, and complications of cardiactransplantation, congenital lung anomalies, atelectasis, pulmonarycongestion and edema, pulmonary embolism, pulmonary hemorrhage,pulmonary infarction, pulmonary hypertension, vascular sclerosis,obstructive pulmonary disease, restrictive pulmonary disease, chronicobstructive pulmonary disease, emphysema, chronic bronchitis, bronchialasthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmalpneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitialdiseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis,desquamative interstitial pneumonitis, hypersensitivity pneumonitis,pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia,diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes,idiopathic pulmonary hemosiderosis, pulmonary involvement incollagen-vascular disorders, pulmonary alveolar proteinosis, lungtumors, inflammatory and noninflammatory pleural effusions,pneumothorax, pleural tumors, drug-induced lung disease,radiation-induced lung disease, and complications of lungtransplantation; an eye disorder such as ocular hypertension andglaucoma; a disorder of cell proliferation such as actinic keratosis,arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixedconnective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnalhemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia;and a cancer, including adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus. Polynucleotidesencoding ENZM may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; in dipstick,pin, and multiformat ELISA-like assays; and in microarrays utilizingfluids or tissues from patients to detect altered ENZM expression. Suchqualitative or quantitative methods are well known in the art.

In a particular embodiment, polynucleotides encoding ENZM may be used inassays that detect the presence of associated disorders, particularlythose mentioned above. Polynucleotides complementary to sequencesencoding ENZM may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantified and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of polynucleotides encoding ENZM in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of ENZM, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding ENZM, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding ENZM may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding ENZM, or a fragment of a polynucleotide complementary to thepolynucleotide encoding ENZM, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived frompolynucleotides encoding ENZM may be used to detect single nucleotidepolymorphisms (SNPs). SNPs are substitutions, insertions and deletionsthat are a frequent cause of inherited or acquired genetic disease inhumans. Methods of SNP detection include, but are not limited to,single-stranded conformation polymorphism (SSCP) and fluorescent SSCP(fSSCP) methods. In SSCP, oligonucleotide primers derived frompolynucleotides encoding ENZM are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

SNPs may be used to study the genetic basis of human disease. Forexample, at least 16 common SNPs have been associated withnon-insulin-dependent diabetes mellitus. SNPs are also useful forexamining differences in disease outcomes in monogenic disorders, suchas cystic fibrosis, sickle cell anemia, or chronic granulomatousdisease. For example, variants in the mannose-binding lectin, MBL2, havebeen shown to be correlated with deleterious pulmonary outcomes incystic fibrosis. SNPs also have utility in pharmacogenomics, theidentification of genetic variants that influence a patient's responseto a drug, such as life-threatening toxicity. For example, a variationin N-acetyl transferase is associated with a high incidence ofperipheral neuropathy in response to the anti-tuberculosis drugisoniazid, while a variation in the core promoter of the ALOX5 generesults in diminished clinical response to treatment with an anti-asthmadrug that targets the 5-lipoxygenase pathway. Analysis of thedistribution of SNPs in different populations is useful forinvestigating genetic drift, mutation, recombination, and selection, aswell as for tracing the origins of populations and their migrations(Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P. Y. andZ. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr.Opin. Neurobiol. 11:637-641).

Methods which may also be used to quantify the expression of ENZMinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves(Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation ofmultiple samples may be accelerated by running the assay in ahigh-throughput format where the oligomer or polynucleotide of interestis presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotides described herein may be used as elementson a microarray. The microarray can be used in transcript imagingtechniques which monitor the relative expression levels of large numbersof genes simultaneously as described below. The rnicroarray may also beused to identify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, ENZM, fragments of ENZM, or antibodies specificfor ENZM may be used as elements on a microarray. The microarray may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time(Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No.5,840,484; hereby expressly incorporated by reference herein). Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467471). Ifa test compound has a signature similar to that of a compound with knowntoxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction of toxicity(see, for example, Press Release 00-02 from the National Institute ofEnvironmental Health Sciences, released Feb. 29, 2000, available athttp://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it isimportant and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In an embodiment, the toxicity of a test compound can be assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosedherein to analyze the proteome of a tissue or cell type. The termproteome refers to the global pattern of protein expression in aparticular tissue or cell type. Each protein component of a proteome canbe subjected individually to further analysis. Proteome expressionpatterns, or profiles, are analyzed by quantifying the number ofexpressed proteins and their relative abundance under given conditionsand at a given time. A profile of a cell's proteome may thus begenerated by separating and analyzing the polypeptides of a particulartissue or cell type. In one embodiment, the separation is achieved usingtwo-dimensional gel electrophoresis, in which proteins from a sample areseparated by isoelectric focusing in the first dimension, and thenaccording to molecular weight by sodium dodecyl sulfate slab gelelectrophoresis in the second dimension (Steiner and Anderson, supra).The proteins are visualized in the gel as discrete and uniquelypositioned spots, typically by staining the gel with an agent such asCoomassie Blue or silver or fluorescent stains. The optical density ofeach protein spot is generally proportional to the level of the proteinin the sample. The optical densities of equivalently positioned proteinspots from different samples, for example, from biological sampleseither treated or untreated with a test compound or therapeutic agent,are compared to identify any changes in protein spot density related tothe treatment. The proteins in the spots are partially sequenced using,for example, standard methods employing chemical or enzymatic cleavagefollowed by mass spectrometry. The identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least5 contiguous amino acid residues, to the polypeptide sequences ofinterest. In some cases, further sequence data may be obtained fordefinitive protein identification.

A proteomic profile may also be generated using antibodies specific forENZM to quantify the levels of ENZM expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena,M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619;Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. etal. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat.No. 5,605,662). Various types of microarrays are well known andthoroughly described in Schena, M., ed. (1999; DNA Microarrays: APractical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encodingENZM may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat.Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acidsequences may be used to develop genetic linkage maps, for example,which correlate the inheritance of a disease state with the inheritanceof a particular chromosome region or restriction fragment lengthpolymorphism (RFLP) (Lander, E. S. and D. Botstein (1986) Proc. Natl.Acad. Sci. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers,supra, pp. 965-968). Examples of genetic map data can be found invarious scientific journals or at the Online Mendelian Inheritance inMan (OMIM) World Wide Web site. Correlation between the location of thegene encoding ENZM on a physical map and a specific disorder, or apredisposition to a specific disorder, may help define the region of DNAassociated with that disorder and thus may further positional cloningefforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation (Gatti, R. A. et al. (1988) Nature 336:577-580). Thenucleotide sequence of the instant invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, ENZM, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between ENZMand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest (Geysen, et al. (1984) PCT application WO84/03564). In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate. The test compounds are reacted with ENZM, orfragments thereof, and washed. Bound ENZM is then detected by methodswell known in the art. Purified ENZM can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding ENZM specificallycompete with a test compound for binding ENZM. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with ENZM.

In additional embodiments, the nucleotide sequences which encode ENZMmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentionedabove and below, including U.S. Ser. No. 60/326,388, U.S. Ser. No.60/328,979, U.S. Ser. No. 60/346,034, U.S. Ser. No. 60/348,284, U.S.Ser. No. 60/338,048, U.S. Ser. No. 60/332,340, U.S. Ser. No. 60/340,357,U.S. Ser. No. 60/387,119, U.S. Ser. No. 60/368,799, U.S. Ser. No.60/368,722, U.S. Ser. No. 60/390,662, and U.S. Ser. No. 60/381,558, arehereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIESEQGOLD database (Incyte Genomics, Palo Alto Calif.). Some tissues werehomogenized and lysed in guanidinium isothiocyanate, while others werehomogenized and lysed in phenol or in a suitable mixture of denaturants,such as TRIZOL (Invitrogen), a monophasic solution of phenol andguanidine isothiocyanate. The resulting lysates were centrifuged overCsCl cushions or extracted with chloroform RNA was precipitated from thelysates with either isopropanol or sodium acetate and ethanol, or byother routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+ RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Invitrogen), using the recommendedprocedures or similar methods known in the art (Ausubel et al., supra,ch. 5). Reverse transcription was initiated using oligo d(T) or randomprimers. Synthetic oligonucleotide adapters were ligated to doublestranded cDNA, and the cDNA was digested with the appropriaterestriction enzyme or enzymes. For most libraries, the cDNA wassize-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, orSEPHAROSE CL4B column chromatography (Amersham Biosciences) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid(Invitrogen, Carlsbad Calif.), PCDNA2.1 plasmid (Invitrogen), PBK-CMVplasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICISplasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto Calif.), pRARE(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.Recombinant plasmids were transformed into competent E. coli cellsincluding XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B,or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN 11 fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamBiosciences or supplied in ABI sequencing kits such as the ABI PRISMBIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (Ausubel et al., supra, ch. 7). Someof the cDNA sequences were selected for extension using the techniquesdisclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM;PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus,Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, PaloAlto Calif.); hidden Markov model (HMM)-based protein family databasessuch as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic AcidsRes. 29:4143); and HMM-based protein domain databases such as SMART(Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is aprobabilistic approach which analyzes consensus primary structures ofgene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct.Biol. 6:361-365.) The queries were performed using programs based onBLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences wereassembled to produce full length polynucleotide sequences.Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,stretched sequences, or Genscan-predicted coding sequences (see ExamplesIV and V) were used to extend Incyte cDNA assemblages to full length.Assembly was performed using programs based on Phred, Phrap, and Consed,and cDNA assemblages were screened for open reading frames usingprograms based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length polypeptide sequences. Alternatively, a polypeptide maybegin at any of the methionine residues of the full length translatedpolypeptide. Full length polypeptide sequences were subsequentlyanalyzed by querying against databases such as the GenBank proteindatabases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, hidden Markov model (BHM)-based protein familydatabases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domaindatabases such as SMART. Full length polynucleotide sequences are alsoanalyzed using MACDNASIS PRO software (MiraiBio, Alameda Calif.) andLASERGENE software (DNASTAR). Polynucleotide and polypeptide sequencealignments are generated using default parameters specified by theCLUSTAL algorithm as incorporated into the MEGALIGN multisequencealignment program (DNASTAR), which also calculates the percent identitybetween aligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:54-106.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative enzymes were initially identified by running the Genscan geneidentification program against public genomic sequence databases (e.g.,gbpri and gbhtg). Genscan is a general-purpose gene identificationprogram which analyzes genomic DNA sequences from a variety of organisms(Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. andS. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The programconcatenates predicted exons to form an assembled cDNA sequenceextending from a methionine to a stop codon. The output of Genscan is aFASTA database of polynucleotide and polypeptide sequences. The maximumrange of sequence for Genscan to analyze at once was set to 30 kb. Todetermine which of these Genscan predicted cDNA sequences encodeenzymes, the encoded polypeptides were analyzed by querying against PFAMmodels for enzymes. Potential enzymes were also identified by homologyto Incyte cDNA sequences that had been annotated as enzymes. Theseselected Genscan-predicted sequences were then compared by BLASTanalysis to the genpept and gbpri public databases. Where necessary, theGenscan-predicted sequences were then edited by comparison to the topBLAST hit from genpept to correct errors in the sequence predicted byGenscan, such as extra or omitted exons. BLAST analysis was also used tofind any Incyte cDNA or public cDNA coverage of the Genscan-predictedsequences, thus providing evidence for transcription. When Incyte cDNAcoverage was available, this information was used to correct or confirmthe Genscan predicted sequence. Full length polynucleotide sequenceswere obtained by assembling Genscan-predicted coding sequences withIncyte cDNA sequences and/or public cDNA sequences using the assemblyprocess described in Example III. Alternatively, full lengthpolynucleotide sequences were derived entirely from edited or uneditedGenscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data “Stitched”Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of ENZM Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:54-106 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm. Sequences from these databases that matched SEQ ID NO:54-106were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Genethon were used to determine if any of theclustered sequences had been previously mapped. Inclusion of a mappedsequence in a cluster resulted in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Genethon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

Association of ENZM Polynucleotides with Parkinson's Disease

Several genes have been identified as showing linkage to autosomaldominant forms of Parkinson's Disease (PD). PD is a commonneurodegenerative disorder causing bradykinesia, resting tremor,muscular rigidity, and postural instability. Cytoplasmic eosinophilicinclusions called Lewy bodies, and neuronal loss especially in thesubstantia nigra pars compacta, are pathological hallmarks of PD(Valente, E. M. et al (2001) Am. J. Hum. Genet. 68:895-900). Lewy bodyParkinson disease has been thought to be a specific autosomal dominantdisorder (Wakabayashi, K. et al. (1998) Acta Neuropath. 96:207-210).Juvenile parkinsonism may be a specific autosomal recessive disorder(Matsumine, H. et al. (1997) Am. J. Hum. Genet. 60:588-596, 1997).(Online Mendelian Inheritance in Man, OMIM. Johns Hopkins University,Baltimore, Md. MIM Number: 168600: Sep. 9, 2002: World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/)

Association of a disease with a chromosomal locus can be determined bylod score. Lod score is a statistical method used to test the linkage oftwo or more loci within families having a genetic disease. The lod scoreis the logarithm to base 10 of the odds in favor of linkage. Linkage isdefined as the tendency of two genes located on the same chromosome tobe inherited together through meiosis (Genetics in Medicine, FifthEdition, (1991) Thompson, M. W. Et al. W.B. Saunders Co. Philadelphia).A lod score of +3 or greater (1000:1 odds in favor of linkage) indicatesa probability of 1 in 1000 that a particular marker was found solely bychance in affected individuals, which is strong evidence that twogenetic loci are linked.

One such gene implicated in PD is PARK3, which maps to 2p13 (Gasser, T.et al. (1998) Nature Genet. 18:262-265). A marker at chromosomalposition D2S441 was found to have a lod score of 3.2 in the region ofPARK3. This marker supported the disease association of PARK3 in thechromosomal interval from D2S134 to D2S286 (Gasser et al., supra).Markers located within chromosomal intervals D2S134 and D2S286, whichmap between 83.88 to 94.05 centiMorgans on the short arm of chromosome2, were used to identify genes that map in the region between D2S134 andD2S286.

A second PD gene, implicated in early-onset recessive parkinsonism, isPARK6, located on chromosome 1 at 1p35-1p36. Several markers wereobtained with lod scores greater than 3 including D1S199, D1S2732,D1S2828, D1S478, D1S2702, D1S2734, D1S2674 (Valente, E. M. et al.supra). These markers were used to determine the PD-relevant range ofchromosome loci and identify sequences that map to chromosome 1 betweenD1S199 and D1S2885. ENZM polynucleotides were found to map within thechromosomal region in which markers associated with disease or otherphysiological processes of interest were located.

Restriction fragment length polymorphism (RFLP) markers shown to be nearregions of DNA known as sequence-tagged sites (STS), have been mapped toNT_Contigs generated by the Human Genome Project using ePCR (Schuler, G.D. (1997) Genome Research 7: 541-550, and (1998) Trends Biotechnol.16(11):456-9). Contigs containing regions of DNA with knowndisease-associated markers are therefore used to identify ENZM sequencesthat map to disease-associated regions of the genome. Contigs longerthan 1 Mb were broken into subcontigs of 1 Mb in length with overlappingsections of 100 kb. A preliminary step used an algorithm, similar toMEGABLAST, to define the mRNA sequence/masked genomic DNA contigpairings. The cDNA/genomic pairings identified by the first algorithmwere confirmed, and the ENZM polynucleotides mapped to DNA contigs,using Sim4 (Florea, L. et al. (1998) Genome Res. 8:967-74, version May2000) which had been optimized in house for high throughput and strandassignment confidence). The SIM4-selected mRNA sequence/genomic contigpairs were further processed to determine the correct location of theENZM polynucleotides on the genomic contig and their strand identity.

SEQ ID NO:7500114 mapped to a region of contig GBI:NT_(—)004359_(—)002.8 from the Feb. 2, 2002 release of NCBI., localizing SEQ IDNO:7500114 to within 14.8 MB of the Parkinson's disease locus onchromosome 6, a chromosomal region consistently associated withParkinson's disease.

Association of ENZM Polynucleotides with Alzheimer's Disease

Restriction fragment length polymorphism (RFLP) markers shown to be nearregions of DNA known as sequence-tagged sites (STS), have been mapped toNT_Contigs generated by the Human Genome Project using ePCR (Schuler, G.D. (1997) Genome Research 7: 541-550, and (1998) Trends Biotechnol.16(11):456-9). Contigs containing regions of DNA with knowndisease-associated markers are therefore used to identify ENZM sequencesthat map to disease-associated regions of the genome. Contigs longerthan 1 Mb were broken into subcontigs of 1 Mb in length with overlappingsections of 100 kb. A preliminary step used an algorithm, similar toMEGABLAST, to define the mRNA sequence/masked genomic DNA contigpairings. The cDNA/genomic pairings identified by the first algorithmwere confirmed, and the ENZM polynucleotides mapped to DNA contigs,using Sim4 (Florea, L. et al. (1998) Genome Res. 8:967-74, version May2000) which had been optimized in house for high throughput and strandassignment confidence). The Sim4 output of the mRNA sequence/genomiccontig pairs was further processed to determine the correct location ofthe ENZM polynucleotides on the genomic contig, and also their strandidentity.

Loci on chromosomes that map to regions associated with particulardiseases can be used as markers for these particular diseases. Thesemarkers then can be used to develop diagnostic and therapeutic tools forthese diseases. For example, loci on chromosome 10 are associated withor linked to Alzheimer's disease (AD), a progressive neurodegenerativedisease that represents the most common form of dementia (Ait-Ghezala,G. et al. (2002) Neurosci Lett. 325:87-90). AD can be inherited as anautosomal dominant trait. Further, genetic studies have focused onidentification of genes that are potential targets for new treatments orimproved diagnostics. The deposition and aggregation of β-amyloid inspecific regions of the brain are key neuropathological hallmarks of AD.Insulin-degrading enzyme (IDE) can degrade β-amyloid Abraham, R. et al.(2001) Hum. Genet. 109:646-652). The IDE gene has been mapped near anAD-associated locus, 10q23-q25 (Espinosa R. 3^(rd) et al. (1991)Cytogenet. Cell Genet. 57:184-186). Linkage analysis using IDE genemarkers was performed on 1426 subjects from 435 families in which atleast two family members were affected with AD.

A logarithm of the odds ratio for linkage (lod) score of over 3indicates a probability of 1 in 1000 that a particular marker was foundsolely by chance in affected individuals. Significant linkage (lod scoreof 3.3) was reported between the polymorphic marker D10S583, located at115.3 cM on chromosome 10, and AD with age of onset ≧50 years (Betram,L. et al. (2000) Science 290:2302-2303). D10S583 maps 36 kb upstream ofthe IDE gene. Further analysis of this region, however, failed to showassociation of SNPs (single nucleotide polymorphisms) within the IDEgene and flanking regions with late-onset AD (LOAD), in a study of 134Caucasian LOAD cases and 111 matched controls from the United Kingdom(Abraham, R. et al, supra). Thus, although the activity of IDE may notinfluence the susceptibility to LOAD, there is substantial linkage inthe chromosomal region containing the IDE gene, marker D10S583, and AD.The IDE gene and D10S583 both map to contig NT_(—)008769, which containsa region of chromosome 10 that is 9.16 Mb in size.

SEQ ID NO:7503454 mapped to a region of contig GBI:NT_(—)008804_(—)005.8from the Feb. 2, 2002 release of NCBI., localizing SEQ ID NO:7503454 towithin 9.16 Mb of the Alzheimer's disease locus on chromosome 10q. Thus,SEQ ID NO:7503454 is in proximity with loci shown to consistentlyassociate with Alzheimer's disease.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook and Russell, supra, ch. 7;Ausubel et al., supra, ch. 4).

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in databases such as GenBank or LIFESEQ(Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{{BLAST}\quad{Score} \times {Percent}\quad{Identity}}{5 \times {minimum}\quad\left\{ {{{length}\quad\left( {{Seq}.\quad 1} \right)},{{length}\quad\left( {{Seq}.\quad 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotides encoding ENZM are analyzed with respectto the tissue sources from which they were derived. For example, somefull length sequences are assembled, at least in part, with overlappingIncyte cDNA sequences (see Example III). Each cDNA sequence is derivedfrom a cDNA library constructed from a human tissue. Each human tissueis classified into one of the following organ/tissue categories:cardiovascular system; connective tissue; digestive system; embryonicstructures; endocrine system; exocrine glands; genitalia, female;genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding ENZM. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of ENZM Encoding Polynucleotides

Full length polynucleotides are produced by extension of an appropriatefragment of the full length molecule using oligonucleotide primersdesigned from this fragment. One primer was synthesized to initiate 5′extension of the known fragment, and the other primer was synthesized toinitiate 3′ extension of the known fragment. The initial primers weredesigned using OLIGO 4.06 software (National Biosciences), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (AmershamBiosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Biosciences). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) tofill-in restriction site overhangs, and transfected into competent E.coli cells. Transformed cells were selected on antibiotic-containingmedia, and individual colonies were picked and cultured overnight at 37°C. in 384-well plates in LB/2× carb liquid media.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene)with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3,and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C.DNA was quantified by PICOGREEN reagent (Molecular Probes) as describedabove. Samples with low DNA recoveries were reamplified using the sameconditions as described above. Samples were diluted with 20%dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energytransfer sequencing primers and the DYENAMIC DIRECT kit (AmershamBiosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the aboveprocedure or are used to obtain 5′ regulatory sequences using the aboveprocedure along with oligonucleotides designed for such extension, andan appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in ENZM EncodingPolynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms(SNPs) were identified in SEQ ID NO:54-106 using the LIFESEQ database(Incyte Genomics). Sequences from the same gene were clustered togetherand assembled as described in Example III, allowing the identificationof all sequence variants in the gene. An algorithm consisting of aseries of filters was used to distinguish SNPs from other sequencevariants. Preliminary filters removed the majority of basecall errors byrequiring a minimum Phred quality score of 15, and removed sequencealignment errors and errors resulting from improper trimming of vectorsequences, chimeras, and splice variants. An automated procedure ofadvanced chromosome analysis analysed the original chromatogram files inthe vicinity of the putative SNP. Clone error filters used statisticallygenerated algorithms to identify errors introduced during laboratoryprocessing, such as those caused by reverse transcriptase, polymerase,or somatic mutation. Clustering error filters used statisticallygenerated algorithms to identify errors resulting from clustering ofclose homologs or pseudogenes, or due to contamination by non-humansequences. A final set of filters removed duplicates and SNPs found inimmunoglobulins or T-cell receptors.

Certain SNPs were selected for further characterization by massspectrometry using the high throughput MASSARRAY system (Sequenom, Inc.)to analyze allele frequencies at the SNP sites in four different humanpopulations. The Caucasian population comprised 92 individuals (46 male,46 female), including 83 from Utah, four French, three Venezuelan, andtwo Amish individuals. The African population comprised 194 individuals(97 male, 97 female), all African Americans. The Hispanic populationcomprised 324 individuals (162 male, 162 female), all Mexican Hispanic.The Asian population comprised 126 individuals (64 male, 62 female) witha reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean,5% Vietnamese, and 8% other Asian. Allele frequencies were firstanalyzed in the Caucasian population; in some cases those SNPs whichshowed no allelic variance in this population were not further tested inthe other three populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:54-106 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Biosciences), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Biosciences). An aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

XI. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting; see, e.g., Baldeschweiler et al., supra), mechanicalmicrospotting technologies, and derivatives thereof. The substrate ineach of the aforementioned technologies should be uniform and solid witha non-porous surface (Schena, M., ed. (1999) DNA Microarrays: APractical Approach, Oxford University Press, London). Suggestedsubstrates include silicon, silica, glass slides, glass chips, andsilicon wafers. Alternatively, a procedure analogous to a dot or slotblot may also be used to arrange and link elements to the surface of asubstrate using thermal, UV, chemical, or mechanical bonding procedures.A typical array may be produced using available methods and machineswell known to those of ordinary skill in the art and may contain anyappropriate number of elements (Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall,A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM DATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Biosciences). The reverse transcription reaction is performedin a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits(Incyte Genomics). Specific control poly(A)⁺ RNAs are synthesized by invitro transcription from non-coding yeast genomic DNA. After incubationat 37° C. for 2 hr, each reaction sample (one with Cy3 and another withCy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide andincubated for 20 minutes at 85° C. to the stop the reaction and degradethe RNA. Samples are purified using two successive CHROMA SPIN 30 gelfiltration spin columns (Clontech, Palo Alto Calif.) and aftercombining, both reaction samples are ethanol precipitated using 1 ml ofglycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.The sample is then dried to completion using a SpeedVAC (SavantInstruments Inc., Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2%SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Biosciences).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte Genomics). Array elements that exhibit at leastabout a two-fold change in expression, a signal-to-background ratio ofat least about 2.5, and an element spot size of at least about 40%, areconsidered to be differentially expressed.

Expression

SEQ ID NO:157, SEQ ID NO:58, and SEQ ID NO:65 showed differentialexpression in breast cancer tissue, as compared to normal breast tissue,as determined by microarray analysis. Histological and molecularevaluation of breast tumors has revealed that the development of breastcancer evolves through a multi-step process whereby pre-malignantmammary epithelial cells undergo a relatively defined sequence of eventsleading to tumor formation. Early in tumor development ductalhyperplasia is observed. Cells undergoing rapid neoplastic growthgradually progress to invasive carcinoma and become metastatic to thelung, bone and potentially other organs. Several factors, ranging from,but not limited to, environmental to genetic, influence tumorprogression and malignant transformation.

In order to better determine the molecular and phenotypiccharacteristics associated with different stages of breast cancer,breast carcinoma cell lines at various stages of tumor progression werecompared to primary human breast epithelial cells. The expression of SEQID NO:57 and SEQ ID NO:58 was increased by at least two-fold in thehuman breast carcinoma line SK-BR-3, isolated from a pleural effusion ofa 43-year-old female, that forms poorly differentiated adenocarcinomawhen injected into nude mice. In contrast, SEQ ID NO:65 expression wasdecreased by at least two-fold in this same line, as compared to breastprimary epithelial HMEC cells. Expression of SEQ ID NO :65 was alsodecreased by at least two-fold in the breast ductal carcinoma linesT-47D and MDA-mb-435S. T-47D is derived from a pleural effusion obtainedfrom a 54-year-old female with infiltrating ductal carcinoma.MDA-mb-435S is a spindle shaped line that evolved from the parent line(435) as isolated by R. Cailleau from the pleural effusion of a31-year-old female with metastatic, ductal carcinoma of the breast.

Further cross comparison of breast cell lines to the non-malignant cellline MCF-10A, isolated from a 36-year-old woman with fibrocysticdisease, was carried out. The expression of SEQ ID NO:57 and SEQ IDNO:58 was decreased by at least two-fold in HMEC, MCF7, T-47D, andMDA-mb-231 cell lines. In addition, SEQ ID NO:57 and SEQ ID NO:58 showeddecreased expression in BT20 as well as all the above cells lines underserum-free growth conditions. MCF7 is a non-malignant adenocarcinomacell line, isolated from the pleural effusion of a 69-year-old female,that retains characteristics of mammary epithelium such as the abilityto process estradiol via cytoplasmic estrogen receptors. BT20 is abreast carcinoma line derived in vitro from cells migrating out of thinslices of a tumor mass from a 74-year-old female. MDA-mb-231 is a breasttumor cell line isolated from the pleural effusion of a 51-year-oldfemale, that forms poorly differentiated adenocarcinoma in nude mice andALS-treated BALB/c mice. The breast primary epithelial line HMEC and thebreast ductal carcinoma line T-47D were described above.

SEQ ID NO:57 and SEQ ID NO:58 were differentially expressed in threeother types of cancer tissues: colon cancer (soft tissue sarcoma),ovarian cancer and prostate cancer, as determined by microarrayanalysis. Soft tissue sarcomas are relatively rare but more than 50% ofnew patients diagnosed with the disease die from it. The molecularpathways leading to the development of sarcoma are relatively unknown.In order to delineate the pathways that might lead to sarcoma formation,a pair comparison of normal and tumor tissue was made with samples froma single donor. SEQ ID NO:57 and SEQ ID NO:58 expression was decreasedby at least two fold in sigmoid colon tumor tissue isolated from a48-year-old female, as compared to normal sigmoid colon tissue. Thecolon tumor originated from a metastatic gastric sarcoma. Ovarian canceris the leading cause of death from a gynecological cancer. The majorityof ovarian cancers are derived from epithelial cells, and 70% ofpatients with epithelial ovarian cancer present with late-stage disease.The expression of SEQ ID NO:57 and SEQ ID NO:58 was increased by atleast two-fold in ovarian adenocarcinoma tissue from a 79-year-oldfemale, as compared to normal ovary tissue from the same donor.

As with most tumors, prostate cancer develops through a multistageprocess ultimately resulting in an aggressive tumor phenotype.Androgen-responsive cells become hyperplastic and evolve intoearly-stage tumors. Although early-stage tumors are oftenandrogen-sensitive and respond to androgen ablation, a population ofandrogen independent cells evolve from the hyperplastic population.These cells represent a more advanced form of prostate tumor that maybecome invasive and potentially metastasize to the bone, brain or lung.In a cross comparison of prostate tumor cell lines to normal prostateepithelial cells PrEC2, the expression of SEQ ID NO:57 and SEQ ID NO:58was increased at least two-fold in the prostate tumor line DU 145,isolated from a metastatic site in the brain of a 69-year-old male withwidespread metastatic prostate carcinoma. This line has no detectablesensitivity to hormones, it forms colonies in semi-solid medium and isonly weakly positive for acid phosphatase. The differential expressionof these sequences was observed in experiments where DU 145 cells weregrown with or without growth factors and hormones.

In addition to its differential expression in breast cancer tissues, SEQID NO:65 was also differentially expressed in the liver tumor line C3Aupon exposure to gemfibrozil and carboxymethyl cellulose (CMC), asdetermined by microarray analysis. The C3A cell line is a clonalderivative of HepG2, a hepatoma cell line isolated from a 15-year-oldmale with a liver tumor. C3A cells were selected for their strongcontact inhibition growth. Gemfibrozil is a fibric acid antilipemicagent which effectively lowers serum triglycerides and producesfavorable changes in lipoproteins. The effect gemfibrozil on geneexpression in C3A cells was examined in a time dose course experiment,in which cells were exposed to 120, 600, 800 or 1200 μg/ml gemfibrozilfor 3 or 6 hours. The expression of SEQ ID NO:65 was decreased by atleast two-fold in C3A cells treated with gemfibrozil dissolved in CMC atall time points and doses examined, as compared to cells treated onlywith the solvent CMC.

SEQ ID NO:63 and SEQ ID NO:64 showed differentially expressed in lungcancer tissue, as determined by microarray analysis. Lung cancer is theleading cause of cancer death for men and the second leading cause ofcancer death for women in the U.S. Lung cancers are divided into fourhistopathologically distinct groups. Three groups, including squamouscell carcinoma and adenocarcinoma, are classified as non-small cell lungcancers, whereas the fourth group is classified as small cell lungcancer. Collectively the non-small cell lung cancers account for 70% ofall cases. Pair comparisons were performed in which tumor tissue wascompared to normal tissue from the same donor. The expression of SEQ IDNO:63 was increased by at least two-fold in lung squamous cell carcinomatissue, which comprised 50% overt tumor cells, derived from a66-year-old male patient, and in lung adenocarcinoma tissue, whichcomprised over 80% overt tumor cells, derived from a 66-year-old femalepatient. The expression of SEQ ID NO:18 was decreased by at leasttwo-fold in lung squamous cell carcinoma tissue derived from a73-year-old male, which comprised 80% overt tumor cells.

These experiments indicate that SEQ ID NO:57, SEQ ID NO:58, and SEQ IDNO:65 are useful in diagnostic assays for breast cancer and as potentialbiological markers and therapeutic agents in the treatment of breastcancers. In addition, results suggest that SEQ ID NO:57 and SEQ ID NO:58are useful in diagnostic assays for colon and prostate cancer and aspotential biological markers and therapeutic agents in the treatment ofcolon and prostate cancers. Finally, these experiments indicate that SEQID NO:63 and SEQ ID NO:64 are useful in diagnostic assays for lungcancer and as potential biological markers and therapeutic agents in thetreatment of lung cancers.

In an alternative example, SEQ ID NO:67 and SEQ ID NO:68 showeddifferential expression in bone osteosarcoma tissues versus normalosteocytes as determined by microarray analysis. The expression of SEQID NO:67 and SEQ ID NO:68 were increased by at least two fold in boneosteosarcoma tissues relative to normal osteocytes. Therefore, SEQ IDNO:67 and SEQ ID NO:68 are useful as a diagnostic marker or as apotential therapeutic target for bone cancer.

In an alternative example, expression of SEQ ID NO:78 was decreased incolon tumor tissue versus matched normal tissue. Matched normal andtumor samples from the same individual, an 83-year-old female diagnosedwith colon cancer, were compared by competitive hybridization. Sampleswere provided by the Huntsman Cancer Institute. Therefore, SEQ ID NO:78is useful in diagnosis and treatment of cell proliferative disorders.

In another example, expression of SEQ ID NO:78 was increased inperipheral blood mononuclear cells (PBMCs) treated with staphlococcalexotoxin B (SEB) for 72 hours. Human peripheral blood mononuclear cells(PBMCs) contain B lymphocytes, T lymphocytes, NK cells, monocytes,dendritic cells and progenitor cells. PBMCs from 7 healthy volunteerdonors were pooled and stimulated with SEB in vitro. The SEB treatedPBMCs from each donor were compared to PBMCs from the same donor, keptin culture for 24 hours in the absence of SEB. Therefore, SEQ ID NO:78is useful in diagnosis and treatment of autoimmune/inflammatorydisorders.

In another example, expression of SEQ ID NO:78 was increased inadipocytes treated with PPAR-gamma and insulin relative to untreatedadipocytes, during the first week of treatment. Primary preadipocyteswere isolated from adipose tissue of a 36year-old female with body massindex (BMI) 27.7. The preadipocytes were cultured and induced todifferentiate into adipocytes by culturing them in a proprietarydifferentiation medium containing an active component such asproliferator-activated receptor gamma agonists (PPAR-γ agonist) andhuman insulin (Zen-Bio). Human preadipocytes were treated with humaninsulin and PPAR agonist for 3 days and subsequently switched to mediumcontaining insulin only for 5, 9, and 12 more days. Differentiatedadipocytes were compared to untreated preadipocytes maintained inculture in the absence of inducing agents. Therefore, SEQ ID NO:78 isuseful in diagnosis and treatment of metabolic disorders.

In still another example, expression of SEQ ID NO:79 was decreased inHT29 colorectal carcinoma cells treated with 5-aza-2-deoxycytidine. Geneexpression profiles were obtained by comparing normal colon tissue totumorous rectal tissue from the same donor. The donor is a 38-year-oldmale with invasive, poorly differentiated adenocarcinoma with metastasesto 2 out of 13 lymph nodes surveyed (TNM classification: T3, N1, Mx).Samples were provided by the Huntsman Cancer Institute. Therefore, SEQID NO:79 is useful in diagnosis and treatment of cell proliferativedisorders.

In an alternative example, SEQ ID NO:98 was downregulated in coloncancer tissue versus normal colon tissue as determined by microarrayanalysis. Expression of SEQ ID NO:98 was decreased in comparison ofnormal tissue from a donor with diseased tissue from the same donor.Therefore, SEQ ID NO:98 can be used in monitoring treatment of, anddiagnostic assays for, colon cancer.

SEQ ID NO:94 and SEQ ID NO:95 were differentially regulated in C3A cellstreated with gemfibrozil versus untreated C3A cells, as determined bymicroarray analysis. Early confluent C3A cells were treated with variousamounts of Gemfibrozil (120, 600, 800, and 1200 μg/ml) dissolved in CMCfor 1, 3, and 6 hours. Parallel samples of C3A cells were treated with1% CMC only, as a control. Expression of SEQ ID NO:94 and SEQ ID NO:95was decreased in 4 of 12 C3A cell samples treated with gemfibrozil.Expression of SEQ ID NO:34 was increased in C3A cells treated withgemfibrozil. Therefore, SEQ ID NO:94 and SEQ ID NO:95 can be used inmonitoring treatment of, and diagnostic assays for, metabolic,cardiovascular, and liver disorders.

In addition, SEQ ID NO:98 showed tissue-specific expression. RNA samplesisolated from a variety of normal human tissues were compared to acommon reference sample. Tissues contributing to the reference samplewere selected for their ability to provide a complete distribution ofRNA in the human body and include brain (4%), heart (7%), kidney (3%),lung (8%), placenta (46%), small intestine (9%), spleen (3%), stomach(6%), testis (9%), and uterus (5%). The normal tissues assayed wereobtained from at least three different donors. RNA from each donor wasseparately isolated and individually hybridized to the microarray. Sincethese hybridization experiments were conducted using a common referencesample, differential expression values are directly comparable from onetissue to another.

The expression of SEQ ID NO:98 was increased by at least two-fold inliver as compared to the reference sample. Therefore, SEQ ID NO:98 canbe used as a tissue marker for liver.

XII. Complementary Polynucleotides

Sequences complementary to the ENZM-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring ENZM. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of ENZM. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the ENZM-encoding transcript.

XIII. Expression of ENZM

Expression and purification of ENZM is achieved using bacterial orvirus-based expression systems. For expression of ENZM in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express ENZM uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof ENZM in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding ENZM by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945).

In most expression systems, ENZM is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamBiosciences). Following purification, the GST moiety can beproteolytically cleaved from ENZM at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel et al.(supra, ch. 10 and 16). Purified ENZM obtained by these methods can beused directly in the assays shown in Examples XVII, XVIII, and XIX,where applicable.

XIV. Functional Assays

ENZM function is assessed by expressing the sequences encoding ENZM atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1plasmid (Invitrogen), both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, for example, an endothelial or hematopoietic cellline, using either liposome formulations or electroporation. 1-2 μg ofan additional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York N.Y.).

The influence of ENZM on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding ENZMand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humaninmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding ENZM and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XV. Production of ENZM Specific Antibodies

ENZM substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizeanimals (e.g., rabbits, mice, etc.) and to produce antibodies usingstandard protocols.

Alternatively, the ENZM amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art (Ausubel et al.,supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity (Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide and anti-ENZM activity by, forexample, binding the peptide or ENZM to a substrate, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring ENZM Using Specific Antibodies

Naturally occurring or recombinant ENZM is substantially purified byimmunoaffinity chromatography using antibodies specific for ENZM. Animmunoaffinity column is constructed by covalently coupling anti-ENZMantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Biosciences). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing ENZM are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof ENZM (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/ENZM binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and ENZMis collected.

XVII. Identification of Molecules Which Interact with ENZM

ENZM, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133:529-539). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled ENZM, washed, and anywells with labeled ENZM complex are assayed. Data obtained usingdifferent concentrations of ENZM are used to calculate values for thenumber, affinity, and association of ENZM with the candidate molecules.

Alternatively, molecules interacting with ENZM are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989;Nature 340:245-246), or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech).

ENZM may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVIII. Demonstration of ENZM Activity

ENZM activity is demonstrated through a variety of specific enzymeassays; some of which are outlined below.

ENZM oxidoreductase activity is measured by the increase in extinctioncoefficient of NAD(P)H coenzyme at 340 nm for the measurement ofoxidation activity, or the decrease in extinction coefficient of NAD(P)Hcoenzyme at 340 nm for the measurement of reduction activity (Dalziel,K. (1963) J. Biol. Chem. 238:2850-2858). One of three substrates may beused: Asn-βGal, biocytidine, or ubiquinone-10. The respective subunitsof the enzyme reaction, for example, cytochrome c₁-b oxidoreductase andcytochrome c, are reconstituted. The reaction mixture contains a) 1-2mg/ml ENZM; and b) 15 mM substrate, 2.4 mM NAD(P)⁺ in 0.1 M phosphatebuffer, pH 7.1 (oxidation reaction), or 2.0 M NAD(P)H, in 0.1 M Na₂HPO₄buffer, pH 7.4 (reduction reaction); in a total volume of 0.1 ml.Changes in absorbance at 340 nm (A₃₄₀) are measured at 23.5° C. using arecording spectrophotometer (Shimadzu Scientific Instruments, Inc.,Pleasanton, Calif.). The amount of NAD(P)H is stoichiometricallyequivalent to the amount of substrate initially present, and the changein A₃₄₀ is a direct measure of the amount of NAD(P)H produced;ΔA₃₄₀=6620[NADH]. ENZM activity is proportional to the amount of NAD(P)Hpresent in the assay.

Aldo/keto reductase activity of ENZM is proportional to the decrease inabsorbance at 340 nm as NADPH is consumed (or increased absorbance ifNADPH is produced, i.e., if the reverse reaction is monitored). Astandard reaction mixture is 135 mM sodium phosphate buffer (pH 6.2-7.2depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mgENZM and an appropriate level of substrate. The reaction is incubated at30° C. and the reaction is monitored continuously with aspectrophotometer. ENZM activity is calculated as mol NADPH consumed/mgof ENZM.

Acyl-CoA dehydrogenase activity of ENZM is measured using an anaerobicelectron transferring flavoprotein (ETF) assay. The reaction mixturecomprises 50 mM Tris-HCl (pH 8.0), 0.5% glucose, and 50 μM acyl-CoAsubstrate (i.e., isovaleryl-CoA) that is pre-warmed to 32° C. Themixture is depleted of oxygen by repeated exposure to vacuum followed bylayering with argon. Trace amounts of oxygen are removed by the additionof glucose oxidase and catalase followed by the addition of ETF to afinal concentration of 1 μM. The reaction is initiated by addition ofpurified ENZM or a sample containing ENZM and exciting the reaction at342 nm. Quenching of fluorescence caused by the transfer of electronsfrom the substrate to ETF is monitored at 496 nm. 1 unit of acyl-CoAdehydrogenase activity is defined as the amount of ENZM required toreduce 1 μmol of ETF per minute (Reinard, T. et al. (2000) J. Biol.Chem. 275:33738-33743).

Alcohol dehydrogenase activity of ENZM is measured by following theconversion of NAD⁺ to NADH at 340 nm (ε₃₄₀=6.22 mM⁻⁴ cm⁻¹) at 25° C. in0.1 M potassium phosphate (pH 7.5), 0.1 M glycine (pH 10.0), and 2.4 mMNAD⁺. Substrate (e.g., ethanol) and ENZM are then added to the reaction.The production of NADH results in an increase in absorbance at 340 nmand correlates with the oxidation of the alcohol substrate and theamount of alcohol dehydrogenase activity in the ENZM sample (Svensson,S. (1999) J. Biol. Chem. 274:29712-29719).

Aldehyde dehydrogenase activity of ENZM is measured by determining thetotal hydrolase+dehydrogenase activity of ENZM and subtracting thehydrolase activity. Hydrolase activity is first determined in a reactionmixture containing 0.05 M Tris-HCl (pH 7.8), 100 mM 2-mercaptoethanol,and 0.5-18 μM substrate, e.g., 10-HCO-HPteGlu(10-formyltetrahydrofolate; HPteGlu, tetrahydrofolate) or 10-FDDF(10-formyl-5,8-dideazafolate). Approximately 1 μg of ENZM is added in afinal volume of 1.0 ml. The reaction is monitored and read against ablank cuvette, containing all components except enzyme. The appearanceof product is measured at either 295 nm for 5,8-dideazafolate or 300 nmfor HPteGlu using molar extinction coefficients of 1.89×10⁴ and 2.17×10⁴for 5,8-dideazafolate and HPteGlu, respectively. The addition of NADP⁺to the reaction mixture allows the measurement of both dehydrogenase andhydrolase activity (assays are performed as before). Based on theproduction of product in the presence of NADP⁺ and the production ofproduct in the absence of the cofactor, aldehyde dehydrogenase activityis calculated for ENZM. In the alternative, aldehyde dehydrogenaseactivity is assayed using propanal as substrate. The reaction mixturecontains 60 mM sodium pyrophosphate buffer (pH 8.5), 5 mM propanal, 1 mMNADP⁺, and ENZM in a total volume of 1 ml. Activity is determined by theincrease in absorbance at 340 nm, resulting from the generation ofNADPH, and is proportional to the aldehyde dehydrogenase activity in thesample (Krupenko, S. A. et al. (1995) J. Biol. Chem. 270:519-522).

6-phosphogluconate dehydrogenase activity of ENZM is measured byincubating purified ENZM, or a composition comprising ENZM, in 120 mMtriethanolamine (pH 7.5), 0.1 mM EDTA, 0.5 mM NADP⁺, and 10-150 μM6-phosphogluconate as substrate at 20-25° C. The production of NADPH ismeasured fluorimetrically (340 nm excitation, 450 nm emission) and isindicative of 6-phosphogluconate dehydrogenase activity. Alternatively,the production of NADPH is measured photometrically, based on absorbanceat 340 nm. The molar amount of NADPH produced in the reaction isproportional to the 6-phosphogluconate dehydrogenase activity in thesample (Tetaud et al., supra).

Ribonucleotide diphosphate reductase activity of ENZM is determined byincubating purified ENZM, or a composition comprising ENZM, along withdithiothreitol, Mg⁺⁺, and ADP, GDP, CDP, or UDP substrate. The productof the reaction, the corresponding deoxyribonucleotide, is separatedfrom the substrate by thin-layer chromatography. The reaction productscan be distinguished from the reactants based on rates of migration. Theuse of radiolabeled substrates is an alternative for increasing thesensitivity of the assay. The amount of deoxyribonucleotides produced inthe reaction is proportional to the amount of ribonucleotide diphosphatereductase activity in the sample (note that this is true only forpre-steady state kinetic analysis of ribonucleotide diphosphatereductase activity, as the enzyme is subject to negative feedbackinhibition by products) (Nutter and Cheng, supra).

Dihydrodiol dehydrogenase activity of ENZM is measured by incubatingpurified ENZM, or a composition comprising ENZM, in a reaction mixturecomprising 50 mM glycine (pH 9.0), 2.3 mM NADP⁺, 8% DMSO, and atrans-dihydrodiol substrate, selected from the group including but notlimited to, (±)-trans-naphthalene-1,2-dihydrodiol,(±)-trans-phenanthrene-1,2-dihydrodiol, and(±)-trans-chrysene-1,2-dihydrodiol. The oxidation reaction is monitoredat 340 nm to detect the formation of NADPH, which is indicative of theoxidation of the substrate. The reaction mixture can also be analyzedbefore and after the addition of ENZM by circular dichroism to determinethe stereochemistry of the reaction components and determine whichenantiomers of a racemic substrate composition are oxidized by the ENZM(Penning, supra).

Glutathione S-transferase (GST) activity of ENZM is determined bymeasuring the ENZM catalyzed conjugation of GSH with1-chloro-2,4-dinitrobenzene (CDNB), a common substrate for most GSTs.ENZM is incubated with 1 mM CDNB and 2.5 mM GSH together in 0.1Mpotassium phosphate buffer, pH 6.5, at 25° C. The conjugation reactionis measured by the change in absorbance at 340 nm using an ultravioletspectrophometer. ENZM activity is proportional to the change inabsorbance at 340 nm.

15-oxoprostaglandin 13-reductase (PGR) activity of ENZM is measuredfollowing the separation of contaminating 15-hydroxyprostaglandindehydrogenase (15-PGDH) activity by DEAE chromatography. Followingisolation of PGR containing fractions (or using the purified ENZM),activity is assayed in a reaction comprising 0.1 M sodium phosphate (pH7.4), 1 mM 2-mercaptoethanol, 20 μg substrate (e.g., 15-oxo derivativesof prostaglandins PGE₁, PGE₂, and PGE_(2α)), and 1 mM NADH (or a higherconcentration of NADPH). ENZM is added to the reaction which is thenincubated for 10 min at 37° C. before termination by the addition of0.25 ml 2 N NaOH. The amount of 15-oxo compound remaining in the sampleis determined by measuring the maximum absorption at 500 nm of theterminated reaction and comparing this value to that of a terminatedcontrol reaction that received no ENZM. 1 unit of enzyme is defined asthe amount required to catalyze the oxidation of 1 μmol substrate perminute and is proportional to the amount of PGR activity in the sample.

Choline dehydrogenase activity of ENZM is identified by the ability ofE. coli, transformed with an ENZM expression vector, to grow on mediacontaining choline as the sole carbon and nitrogen source. The abilityof the transformed bacteria to thrive is indicative of cholinedehydrogenase activity (Magne Østerås, M. (1998) Proc. Natl. Acad. Sci.USA 95:11394-11399).

ENZM thioredoxin activity is assayed as described (Luthman, M. (1982)Biochemistry 21:6628-6633). Thioredoxins catalyze the formation ofdisulfide bonds and regulate the redox environment in cells to enablethe necessary thiol:disulfide exchanges. One way to measure thethiol:disulfide exchange is by measuring the reduction of insulin in amixture containing 0.1 M potassium phosphate, pH 7.0, 2 mM EDTA, 0.16 μMinsulin, 0.33 mM DTT, and 0.48 mM NADPH. Different concentrations ofENZM are added to the mixture, and the reaction rate is followed bymonitoring the oxidation of NADPH at 340 nM.

ENZM transferase activity is measured through assays such as a methyltransferase assay in which the transfer of radiolabeled methyl groupsbetween a donor substrate and an acceptor substrate is measured (Bokar,J. A. et al. (1994) J. Biol. Chem. 269:17697-17704). Reaction mixtures(50 μl final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mMdithiothreitol, 3% polyvinylalcohol, 1.5 μCi [methyl-³H]AdoMet (0.375 μMAdoMet) (DuPont-NEN), 0.6 μg ENZM, and acceptor substrate (0.4 μg[³⁵S]RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration).Reaction mixtures are incubated at 30° C. for 30 minutes, then at 65° C.for 5 minutes. The products are separated by chromatography orelectrophoresis and the level of methyl transferase activity isdetermined by quantification of methyl-³H recovery.

Aminotransferase activity of ENZM is assayed by incubating samplescontaining ENZM for 1 hour at 37° C. in the presence of 1 mML-kynurenine and 1 mM 2-oxoglutarate in a final volume of 200 μl of 150mM Tris acetate buffer (pH 8.0) containing 70 μM PLP. The formation ofkynurenic acid is quantified by HPLC with spectrophotometric detectionat 330 nm using the appropriate standards and controls well known tothose skilled in the art. In the alternative, L-3-hydroxykynurenine isused as substrate and the production of xanthurenic acid is determinedby HPLC analysis of the products with UV detection at 340 nm. Theproduction of kynurenic acid and xanthurenic acid, respectively, isindicative of aminotransferase activity (Buchli et al., supra).

In another alternative, aminotransferase activity of ENZM is measured bydetermining the activity of purified ENZM or crude samples containingENZM toward various amino and oxo acid substrates under single turnoverconditions by monitoring the changes in the UV/VIS absorption spectrumof the enzyme-bound cofactor, pyridoxal 5′-phosphate (PLP). Thereactions are performed at 25° C. in 50 mM 4-methylmorpholine (pH 7.5)containing 9 μM purified ENZM or ENZM containing samples and substrateto be tested (amino and oxo acid substrates). The half-reaction fromamino acid to oxo acid is followed by measuring the decrease inabsorbance at 360 nm and the increase in absorbance at 330 nm due to theconversion of enzyme-bound PLP to pyridoxamine 5′ phosphate (PMP). Thespecificity and relative activity of ENZM is determined by the activityof the enzyme preparation against specific substrates (Vacca, supra).

ENZM chitinase activity is determined with the fluorogenic substrates4-methylumbelliferyl chitotriose, methylumbelliferyl chitobiose, ormethylumbelliferyl N-acetylglucosamine. Purified ENZM is incubated with0.5 uM substrate at pH 4.0 (0.1M citrate buffer), pH 5.0 (0.1M phosphatebuffer), or pH 6.0 (0.1M Tris-HCL). After various times of incubation,the reaction is stopped by the addition of 0.1M glycine buffer, pH 10.4,and the concentration of free methylumbelliferone is determinedfluorometrically. Chitinase B from Serratia marcescens may be used as apositive control (Hakala, supra).

ENZM isomerase activity is determined by measuring2-hydroxyhepta-2,4-diene,1,7 dioate isomerase (HHDD isomerase) activity,as described by Garrido-Peritierra, A. and R. A. Cooper (1981; Eur. J.Biochem. 17:581-584). The sample is combined with5-carboxymethyl-2-oxo-hex-3-ene-1,5, dioate (CMHD), which is thesubstrate for HHDD isomerase. CMHD concentration is monitored bymeasuring its absorbance at 246 nm. Decrease in absorbance at 246 nm isproportional to HHDD isomerase activity of ENZM.

ENZM isomerase activity such as peptidyl prolyl cis/trans isomeraseactivity can be assayed by an enzyme assay described by Rahfeld (supra).The assay is performed at 10° C. in 35 mM HEPES buffer, pH 7.8,containing chymotrypsin (0.5 mg/ml) and ENZM at a variety ofconcentrations. Under these assay conditions, the substrate,Suc-Ala-Xaa-Pro-Phe-4-NA, is in equilibrium with respect to the prolylbond, with 80-95% in trans and 5-20% in cis conformation. An aliquot (2μl) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is addedto the reaction mixture described above. Only the cis isomer is asubstrate for cleavage by chymotrypsin. Thus, as the substrate isisomerized by ENZM, the product is cleaved by chymotrypsin to produce4-nitroanilide, which is detected by its absorbance at 390 nm.4-Nitroanilide appears in a time-dependent and a ENZMconcentration-dependent manner.

Alternatively, peptidyl prolyl cis-trans isomerase activity of ENZM canbe assayed using a chromogenic peptide in a coupled assay withchymotrypsin (Fischer, G. et al. (1984) Biomed. Biochim. Acta43:1101-1111).

UDP glucuronyltransferase activity of ENZM is measured using acolorimetric determination of free amine groups (Gibson, G. G. and P.Skett (1994) Introduction to Drug Metabolism, Blackie Academic andProfessional, London). An amine-containing substrate, such as2-aminophenol, is incubated at 37° C. with an aliquot of the enzyme in areaction buffer containing the necessary cofactors (40 mM Tris pH 8.0,7.5 mM MgCl₂, 0.025% Triton X-100, 1 mM ascorbic acid, 0.75 mMUDP-glucuronic acid). After sufficient time, the reaction is stopped byaddition of ice-cold 20% trichloroacetic acid in 0.1 M phosphate bufferpH 2.7, incubated on ice, and centrifuged to clarify the supernatant.Any unreacted 2-aminophenol is destroyed in this step. Sufficientfreshly-prepared sodium nitrite is then added; this step allowsformation of the diazonium salt of the glucuronidated product. Excessnitrite is removed by addition of sufficient ammonium sulfamate, and thediazonium salt is reacted with an aromatic amine (for example,N-naphthylethylene diamine) to produce a colored azo compound which canbe assayed spectrophotometrically (at 540 nm, for example). A standardcurve can be constructed using known concentrations of aniline, whichwill form a chromophore with similar properties to 2-aminophenolglucuronide.

Adenylosuccinate synthetase activity of ENZM is measured by synthesis ofAMP from IMP. The sample is combined with AMP. IMP concentration ismonitored spectrophotometrically at 248 nm at 23° C. (Wang, W. et al.(1995) J. Biol. Chem. 270:13160-13163). The increase in IMPconcentration is proportional to ENZM activity.

Alternatively, AMP binding activity of ENZM is measured by combining thesample with ³²P-labeled AMP. The reaction is incubated at 37° C. andterminated by addition of trichloroacetic acid. The acid extract isneutralized and subjected to gel electrophoresis to remove unboundlabel. The radioactivity retained in the gel is proportional to ENZMactivity.

In another alternative, xenobiotic carboxylic acid:CoA ligase activityof ENZM is measured by combining the sample with γ⁻³³P-ATP and measuringthe formation of γ-³³P-pyrophosphate with time (Vessey, D. A. et al.(1998) . Biochem. Mol. Toxicol. 12:151-155).

Protein phosphatase (PP) activity can be measured by the hydrolysis ofP-nitrophenyl phosphate (PNPP). ENZM is incubated together with PNPP inHEPES buffer pH 7.5, in the presence of 0.1% β-mercaptoethanol at 37° C.for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH(Diamond, R. H. et al. (1994) Mol. Cell. Biol. 14:3752-62).

Alternatively, acid phosphatase activity of ENZM is demonstrated byincubating ENZM containing extract with 100 μl of 10 mM PNPP in 0.1 Msodium citrate, pH 4.5, and 50 μl of 40 mM NaCl at 37° C. for 20 min.The reaction is stopped by the addition of 0.5 ml of 0.4 M glycine/NaOH,pH 10.4 (Saftig, P. et al. (1997) J. Biol. Chem. 272:18628-18635). Theincrease in light absorbance at 410 nm resulting from the hydrolysis ofPNPP is measured using a spectrophotometer. The increase in lightabsorbance is proportional to the activity of ENZM in the assay.

In the alternative, ENZM activity is determined by measuring the amountof phosphate removed from a phosphorylated protein substrate. Reactionsare performed with 2 or 4 nM ENZM in a final volume of 30 μl containing60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% 2-mercaptoethanol and 10μM substrate, ³²P-labeled on serine/threonine or tyrosine, asappropriate. Reactions are initiated with substrate and incubated at 30°C. for 10-15 min. Reactions are quenched with 450 μl of 4% (w/v)activated charcoal in 0.6 M HCl, 90 mM Na₄P₂O₇, and 2 mM NaH₂PO₄, thencentrifuged at 12,000×g for 5 min. Acid-soluble ³²Pi is quantified byliquid scintillation counting (Sinclair, C. et al. (1999) J. Biol. Chem.274:23666-23672).

The adenosine deaminase activity of ENZM is determined by measuring therate of deamination that occurs when adenosine substrate is incubatedwith ENZM. Reactions are performed with a predetermined amount of ENZMin a final volume of 3.0 ml containing 53.3 mM potassium phosphate and0.045 mM adenosine. Assay reagents excluding ENZM are mixed in a quartzcuvette and equilibrated to 25° C. Reactions are initiated by theaddition of ENZM and are mixed immediately by inversion. The decrease inlight absorbance at 265 nm resulting from the hydrolysis of adenosine toinosine is measured using a spectrophotometer. The decrease in theA_(265 nm) is recorded for approximately 5 minutes. The decrease inlight absorbance is proportional to the activity of ENZM in the assay.

ENZM hydrolase activity is measured by the hydrolysis of appropriatesynthetic peptide substrates conjugated with various chromogenicmolecules in which the degree of hydrolysis is quantified byspectrophotometric (or fluorometric) absorption of the releasedchromophore (Beynon and Bond, supra, pp. 25-55). Peptide substrates aredesigned according to the category of protease activity as endopeptidase(serine, cysteine, aspartic proteases), aminopeptidase (leucineaminopeptidase), or carboxypeptidase (Carboxypeptidase A and B,procollagen C-proteinase).

An assay for carbonic anhydrase activity of ENZM uses the fluorescent pHindicator 8-hydroxypyrene-1,3,6-trisulfonate (pyranine) in combinationwith stopped-flow fluorometry to measure carbonic anhydrase activity(Shingles, et al. 1997, Anal. Biochem 252:190-197). A pH 6.0 solution ismixed with a pH 8.0 solution and the initial rate of bicarbonatedehydration is measured. Addition of carbonic anhydrase to the pH 6.0solution enables the measurement of the initial rate of activity atphysiological temperatures with resolution times of 2 ms. Shingles etal. (supra) used this assay to resolve differences in activity andsensitivity to sulfonamides by comparing mammalian carbonic anhydraseisoforms. The fluorescent technique's sensitivity allows thedetermination of initial rates with a protein concentration as little as65 ng/ml.

Decarboxylase activity of ENZM is measured as the release of CO₂ fromlabeled substrate. For example, ornithine decarboxylase activity of ENZMis assayed by measuring the release of CO₂ from L-[1-¹⁴C]-ornithine(Reddy, S. G et al. (1996) J. Biol. Chem. 271:24945-24953). Activity ismeasured in 200 μl assay buffer (50 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 2mM dithiothreitol, 5 mM NaF, 0.1% Brij35, 1 mM PMSF, 60 μMpyridoxal-5-phosphate) containing 0.5 mM L-ornithine plus 0.5 μCiL-[1-¹⁴C]ornithine. The reactions are stopped after 15-30 minutes byaddition of 1 M citric acid, and the ¹⁴CO₂ evolved is trapped on a paperdisk filter saturated with 20 μl of 2 N NaOH. The radioactivity on thedisks is determined by liquid scintillation spectography. The amount of¹⁴CO₂ released is proportional to ornithine decarboxylase activity ofENZM.

AdoHCYase activity of ENZM in the hydrolytic direction is performedspectroscopically by measuring the rate of the product (homocysteine)formed by reaction with 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB). To800 μl of an enzyme solution containing 4.7 μg of ENZM and 4 units ofadenosine deaminase in 50 mM potassium phosphate buffer, pH 7.2,containing 1 mM EDTA (buffer A), is added 200 μl ofS-Adenosyl-L-homocysteine (500 μM) containing 250 μM DTNB in buffer A.The reaction mixture is incubated at 37° C. for 2 minutes. Hydrolyticactivity is monitored at 412 nm continuously using a diode array UVspectrophotometer. Enzyme activity is defined as the amount of enzymethat can hydrolyze 1 μmol of S-Adenosyl-L-homocysteine/minute (Yuan, C-Set al. (1996) J. Biol. Chem. 271:28009-28015).

AdoHCYase activity of ENZM can be measured in the synthetic direction asthe production of S-adenosyl homocysteine using 3-deazaadenosine as asubstrate (Sganga et al. supra). Briefly, ENZM is incubated in a 100 μlvolume containing 0.1 mM 3-deazaadenosine, 5 mM homocysteine, 20 mMHEPES (pH 7.2). The assay mixture is incubated at 37° C. for 15 minutes.The reaction is terminated by the addition of 10 μl of 3 M perchloricacid. After incubation on ice for 15 minutes, the mixture is centrifugedfor 5 minutes at 18,000×g in a microcentrifuge at 4° C. The supernatantis removed, neutralized by the addition of 1 M potassium carbonate, andcentrifuged again. A 50 μl aliquot of supernatant is thenchromatographed on an Altex Ultrasphere ODS column (5 μm particles,4.6×250 mm) by isocratic elution with 0.2 M ammonium dihydrogenphosphate (Aldrich) at a flow rate of 1 ml/min. Protein is determined bythe bicinchrominic acid assay (Pierce).

Alternatively, AdoHCYase activity of ENZM can be measured in thesynthetic direction by a TLC method (Hershfield, M. S. et al. (1979) J.Biol. Chem. 254:22-25). In a preincubation step, 50 μM [8⁻¹⁴C]adenosineis incubated with 5 molar equivalents of NAD⁺ for 15 minutes at 22° C.Assay samples containing ENZM in a 50 μl final volume of 50 mM potassiumphosphate buffer, pH 7.4, 1 mM DTT, and S mM homocysteine, are mixedwith the preincubated [8⁻¹⁴C]adenosine/NAD⁺ to initiate the reaction.The reaction is incubated at 37° C., and 1 μl samples are spotted on TLCplates at 5 minute intervals for 30 minutes. The chromatograms aredeveloped in butanol-1/glacial acetic acid/water (12:3:5, v/v) anddried. Standards are used to identify substrate and products underultraviolet light. The complete spots containing [¹⁴C]adenosine and[¹⁴C]SAH are then detected by exposing x-ray film to the TLC plate. Theradiolabeled substrate and product are then cut from the chromatogramsand counted by liquid scintillation spectrometry. Specific activity ofthe enzyme is determined from the linear least squares slopes of theproduct vs time plots and the milligrams of protein in the sample(Bethin, K. E. et al. (1995) J. Biol. Chem. 270:20698-20702).

Asparaginase activity of ENZM can be measured in the hydrolyticdirection by determining the amount of radiolabeled L-aspartate releasedfrom 0.6 mM N⁴-β′-N-acetylglucosaminyl-L-asparagine substrate when it isincubated at 25° C. with ENZM in 50 mM phosphate buffer, pH 7.5(Kaartinen, V. et al. (1991) J. Biol. Chem. 266:5860-5869).

Acyl CoA Acid Hydrolase activity of ENZM in the hydrolytic direction isperformed spectroscopically by monitoring the appearance of the product(CoASH) formed by reaction of substrate (acylCoA) and ENZM with5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB). The final reaction volume is1 ml of 0.05 M potassium phosphate buffer, pH 8, containing 0.1 mM DTNB,20 μg/ml bovine serum albumin, 10 μM of acyl-CoA of different lengths(C6-CoA, C10-CoA, C14-CoA and C18-CoA, Sigma), and ENZM. The reactionmixture is incubated at 22° C. for 7 minutes. Hydrolytic activity ismonitored spectrophotometrically by measuring absorbance at 412 nm(Poupon, V. et al. (1999) J. Biol. Chem. 274:19188-19194).

ENZM activity of ENZM can be measured spectrophotometrically bydetermining the amount of solubilized RNA that is produced as a resultof incubation of RNA substrate with ENZM. 5 μl (20 μg) of a 4 mg/mlsolution of yeast tRNA (Sigma) is added to 0.8 ml of 40 mM sodiumphosphate, pH 7.5, containing ENZM. The reaction is incubated at 25° C.for 15 minutes. The reaction is stopped by addition of 0.5 ml of anice-cold fresh solution of 20 mM lanthanum nitrate plus 3% perchloricacid. The stopped reaction is incubated on ice for at least 15 min, andthe insoluble tRNA is removed by centrifugation for 5 min at 10,000 g.Solubilized tRNA is determined as UV absorbance (260 nm) of theremaining supernatant, with A₂₆₀ of 1.0 corresponding to 40 μg ofsolubilized RNA (Rosenberg, H. F. et al. (1996) Nucleic Acids Research24:3507-3513).

ENZM activity can be determined as the ability of ENZM to cleave ³²Pinternally labeled T. thermophila pre-tRNA^(Gln). ENZM and substrate areadded to reaction vessels and reactions are carried out in MBB buffer(50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂) for 1 hour at 37° C. Reactionsare terminated with the addition of an equal volume of sample loadingbuffer (SLB: 40 mM EDTA, 8 M urea, 0.2% xylene cyanol, and 0.2%bromophenol blue). The reaction products are separated byelectrophoresis on 8 M urea, 6% polyacrylamide gels and analyzed usingdetection instruments and software capable of quantification of theproducts. One unit of ENZM activity is defined as the amount of enzymerequired to cleave 10% of 28 fmol of T. thermophila pre-tRNA^(Gln) tomature products in 1 hour at 37° C. (True, H. L. et al. (1996) J. Biol.Chem. 271:16559-16566).

Alternatively, cleavage of ³²P internally labeled substrate tRNA by ENZMcan be determined in a 20 μl reaction mixture containing 30 mM HEPES-KOH(pH 7.6), 6 mM MgCl₂, 30 mM KCl, 2 mM DTT, 25 μg/ml bovine serumalbumin, 1 unit/μl rRNasin, and 5,000-50,000 cpm of gel-purifiedsubstrate RNA. 3.0 μl of ENZM is added to the reaction mixture, which isthen incubated at 37° C. for 30 minutes. The reaction is stopped byguanidinium/phenol extraction, precipitated with ethanol in the presenceof glycogen, and subjected to denaturing polyacrylamide gelelectrophoresis (6 or 8% polyacrylamide, 7 M urea) and autoradiography(Rossmanith, W. et al. (1995) J. Biol. Chem. 270:12885-12891). The ENZMactivity is proportional to the amount of cleavage products detected.

ENZM activity can be measured by determining the amount of freeadenosine produced by the hydrolysis of AMP, as described by Sala-Newbyet al., supra. Briefly, ENZM is incubated with AMP in a suitable bufferfor 10 minutes at 37° C. Free adenosine is separated from AMP andmeasured by reverse phase HPLC.

Alternatively, ENZM activity is measured by the hydrolysis ofADP-ribosylarginine (Konczalik, P. and J. Moss (1999) J. Biol. Chem.274:16736-16740). 50 ng of ENZM is incubated with 100 μMADP-ribosyl-[¹⁴C]arginine (78,000 cpm) in 50 mM potassium phosphate, pH7.5, 5 mM dithiothreitol, 10 mM MgCl₂ in a final volume of 100 μl. After1 h at 37° C., 90 μl of the sample is applied to a column (0.5×4 cm) ofAffi-Gel 601 (boronate) equilibrated and eluted with five 1-ml portionsof 0.1 M glycine, pH 9.0, 0.1 M NaCl, and 10 mM MgCl₂. Free ¹⁴C-Arg inthe total eluate is measured by liquid scintillation counting.

Epoxide hydrolase activity of ENZM can be determined with a radiometricassay utilizing [H³]-labeled trans-stilbene oxide (TSO) as substrate.Briefly, ENZM is preincubated in Tris-HCl pH 7.4 buffer in a totalvolume of 100 μl for 1 minute at 37° C. 1 μl of [H³]-labeled TSO (0.5 μMin EtOH) is added and the reaction mixture is incubated at 37° C. for 10minutes. The reaction mixture is extracted with 200 μl n-dodecane. 50 μlof the aqueous phase is removed for quantification of diol product in aliquid scintillation counter (LSC). ENZM activity is calculated as nmoldiol product/min/mg protein (Gill, S. S. et al. (1983) AnalyticalBiochemistry 131:273-282).

Lysophosphatidic acid acyltransferase activity of ENZM is measured byincubating samples containing ENZM with 1 mM of the thiol reagent5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 50 μm LPA, and 50 μmacyl-CoA in 100 mM Tris-HCl, pH 7.4. The reaction is initiated byaddition of acyl-CoA, and allowed to reach equilibrium. Transfer of theacyl group from acyl-CoA to LPA releases free CoA, which reacts withDTNB. The product of the reaction between DTNB and free CoA absorbs at413 nm. The change in absorbance at 413 nm is measured using aspectrophotometer, and is proportional to the lysophosphatidic acidacyltransferase activity of ENZM in the sample.

N-acyltransferase activity of ENZM is measured using radiolabeled aminoacid substrates and measuring radiolabel incorporation into conjugatedproducts. ENZM is incubated in a reaction buffer containing an unlabeledacyl-CoA compound and radiolabeled amino acid, and the radiolabeledacyl-conjugates are separated from the unreacted amino acid byextraction into n-butanol or other appropriate organic solvent. Forexample, Johnson, M. R. et al. (1990; J. Biol. Chem. 266:10227-10233)measured bile acid-CoA:amino acid N-acyltransferase activity byincubating the enzyme with cholyl-CoA and ³H-glycine or ³H-taurine,separating the tritiated cholate conjugate by extraction into n-butanol,and measuring the radioactivity in the extracted product byscintillation. Alternatively, N-acyltransferase activity is measuredusing the spectrophotometric determination of reduced CoA (CoASH)described below.

N-acetyltransferase activity of ENZM is measured using the transfer ofradiolabel from [¹⁴C]acetyl-CoA to a substrate molecule (for example,see Deguchi, T. (1975) J. Neurochem. 24:1083-5). Alternatively, a newerspectrophotometric assay based on DTNB reaction with CoASH may be used.Free thiol-containing CoASH is formed during N-acetyltransferasecatalyzed transfer of an acetyl group to a substrate. CoASH is detectedusing the absorbance of DTNB conjugate at 412 nm (De Angelis, J. et al.(1997) J. Biol. Chem. 273:3045-3050). ENZM activity is proportional tothe rate of radioactivity incorporation into substrate, or the rate ofabsorbance increase in the spectrophotometric assay.

Galactosyltransferase activity of ENZM is determined by measuring thetransfer of galactose from UDP-galactose to a GlcNAc-terminatedoligosaccharide chain in a radioactive assay. (Kolbinger, F. et al.(1998) J. Biol. Chem. 273:58-65.) The ENZM sample is incubated with 14μl of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/mlbovine serum albumin, 0.26 mM UDP-galactose, 2 μl of UDP-[³H]galactose),1 μl of MnCl₂ (500 mM), and 2.5 μl of GlcNAcβO—(CH₂)₈—CO₂Me (37 mg/ml indimethyl sulfoxide) for 60 minutes at 37° C. The reaction is quenched bythe addition of 1 ml of water and loaded on a C18 Sep-Pak cartridge(Waters), and the column is washed twice with 5 ml of water to removeunreacted UDP-[³H]galactose. The [³H]galactosylated GlcNAcβO—CH₂)₈—CO₂Meremains bound to the column during the water washes and is eluted with 5ml of methanol. Radioactivity in the eluted material is measured byliquid scintillation counting and is proportional togalactosyltransferase activity of ENZM in the starting sample.

Phosphoribosyltransferase activity of ENZM is measured as the transferof a phosphoribosyl group from phosphoribosylpyrophosphate (PRPP) to apurine or pyridine base. Assay mixture (20 μl) containing 50 mM Trisacetate, pH 9.0, 20 mM 2-mercaptoethanol, 12.5 mM MgCl₂, and 0.1 mMlabeled substrate, for example, [¹⁴C]uracil, is mixed with 20 μl of ENZMdiluted in 0.1 M Tris acetate, pH 9.7, and 1 mg/ml bovine serum albumin.Reactions are preheated for 1 min at 37° C., initiated with 10 μl of 6mM PRPP, and incubated for 5 min at 37° C. The reaction is stopped byheating at 100° C. for 1 min. The product [¹⁴C]UMP is separated from[¹⁴C]uracil on DEAE-cellulose paper (Turner, R. J. et al. (1998) J.Biol. Chem. 273:5932-5938). The amount of [¹⁴C]UMP produced isproportional to the phosphoribosyltransferase activity of ENZM.

ADP-ribosyltransferase activity of ENZM is measured as the transfer ofradiolabel from adenine-NAD to agmatine (Weng, B. et al. (1999) J. Biol.Chem. 274:31797-31803). Purified ENZM is incubated at 30° C. for 1 hr ina total volume of 300 μl containing 50 mM potassium phosphate (pH, 7.5),20 mM agmatine, and 0.1 mM [adenine-U-¹⁴C]NAD (0.05 mCi). Samples (100μl) are applied to Dowex columns and [¹⁴C]ADP-ribosylagmatine elutedwith 5 ml of water for liquid scintillation counting. The amount ofradioactivity recovered is proportional to ADP-ribosyltransferaseactivity of ENZM.

An ENZM activity assay measures aminoacylation of tRNA in the presenceof a radiolabeled substrate. SYNT is incubated with [¹⁴C]-labeled aminoacid and the appropriate cognate tRNA (for example, [¹⁴C]alanine andtRNA^(ala)) in a buffered solution. ¹⁴C-labeled product is separatedfrom free [¹⁴C]amino acid by chromatography, and the incorporated ¹⁴C isquantified using a scintillation counter. The amount of ¹⁴C-labeledproduct detected is proportional to the activity of ENZM in this assay(Ibba, M. et al. (1997) Science 278:1119-1122).

Alternatively, argininosuccinate synthase activity of ENZM is measuredbased on the conversion of [³H]aspartate to [³H]argininosuccinate. ENZMis incubated with a mixture of [³C]aspartate, citruline, Tris-HCl (pH7.5), ATP, MgCl₂, KCl, phosphoenolpyruvate, pyruvate kinase, myokinase,and pyrophosphatase, and allowed to proceed for 60 minutes at 37° C.Enzyme activity was terminated with addition of acetic acid and heatingfor 30 minutes at 90° C. [³H]argininosuccinate is separated fromun-catalyzed [³H]aspartate by chromatography and quantified by liquidscintillation spectrometry. The amount of [³]argininosuccinate detectedis proportional to the activity of ENZM in this assay (O'Brien, W. E.(1979) Biochemistry 18:5353-5356).

Alternatively, the esterase activity of ENZM is assayed by thehydrolysis of p-nitrophenylacetate (NPA). ENZM is incubated togetherwith 0.1 μM NPA in 0.1 M potassium phosphate buffer (pH 7.25) containing150 mM NaCl. The hydrolysis of NPA is measured by the increase ofabsorbance at 400 nm with a spectrophotometer. The increase in lightabsorbance is proportional to the activity of ENZM (Probst, M. R. et al.(1994) J. Biol. Chem. 269:21650-21656).

XIX. Identification of ENZM Agonists and Antagonists

Agonists or antagonists of ENZM activation or inhibition may be testedusing the assays described in section XVIII. Agonists cause an increasein ENZM activity and antagonists cause a decrease in ENZM activity.

Various modifications and variations of the described compositions,methods, and systems of the invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.It will be appreciated that the invention provides novel and usefulproteins, and their encoding polynucleotides, which can be used in thedrug discovery process, as well as methods for using these compositionsfor the detection, diagnosis, and treatment of diseases and conditions.Although the invention has been described in connection with certainembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Nor shouldthe description of such embodiments be considered exhaustive or limitthe invention to the precise forms disclosed. Furthermore, elements fromone embodiment can be readily recombined with elements from one or moreother embodiments. Such combinations can form a number of embodimentswithin the scope of the invention. It is intended that the scope of theinvention be defined by the following claims and their equivalents.TABLE 1 Incyte Polypeptide Incyte Polynucleotide Polynucleotide IncyteProject ID SEQ ID NO: Polypeptide ID SEQ ID NO: ID Incyte Full LengthClones 7499940 1 7499940CD1 54 7499940CB1 90059996CA2 3329870 23329870CD1 55 3329870CB1 7500698 3 7500698CD1 56 7500698CB1 7500223 47500223CD1 57 7500223CB1 7500295 5 7500295CD1 58 7500295CB1 2134968CA27502095 6 7502095CD1 59 7502095CB1 7500507 7 7500507CD1 60 7500507CB190150580CA2 7500840 8 7500840CD1 61 7500840CB1 7493620 9 7493620CD1 627493620CB1 7494697 10 7494697CD1 63 7494697CB1 90156851CA2 8146738 118146738CD1 64 8146738CB1 7500114 12 7500114CD1 65 7500114CB1 6054195CA27500197 13 7500197CD1 66 7500197CB1 7500145 14 7500145CD1 67 7500145CB17500874 15 7500874CD1 68 7500874CB1 7500495 16 7500495CD1 69 7500495CB15723074CA2, 90162244CA2 7500194 17 7500194CD1 70 7500194CB1 7500871 187500871CD1 71 7500871CB1 1486817CA2, 157510CA2, 3737615CA2, 6383983CA2,90156928CA2, 90156955CA2, 90188640CA2, 90188703CA2, 90188732CA2,90188735CA2, 90188920CA2 7500873 19 7500873CD1 72 7500873CB1 1486817CA2,157510CA2, 3737615CA2, 6383983CA2, 90156928CA2, 90156955CA2,90188640CA2, 90188703CA2, 90188732CA2, 90188735CA2, 90188920CA2 750349120 7503491CD1 73 7503491CB1 7503427 21 7503427CD1 74 7503427CB190176824CA2, 90176832CA2 7503547 22 7503547CD1 75 7503547CB1 7975468CA21932641 23 1932641CD1 76 1932641CB1 6892447 24 6892447CD1 77 6892447CB17503416 25 7503416CD1 78 7503416CB1 7503874 26 7503874CD1 79 7503874CB190053561CA2 7503454 27 7503454CD1 80 7503454CB1 90009326CA2, 90177533CA27503528 28 7503528CD1 81 7503528CB1 7503705 29 7503705CD1 82 7503705CB17503707 30 7503707CD1 83 7503707CB1 90001962 31 90001962CD1 8490001962CB1 90001962CA2 70819231 32 70819231CD1 85 70819231CB12967971CA2 7504066 33 7504066CD1 86 7504066CB1 2455713CA2, 90029385CA2,90035649CA2, 90087151CA2, 90137747CA2, 90137824CA2, 90137863CA2,90137879CA2, 90138023CA2, 90138031CA2, 90161864CA2, 90161872CA2,90161880CA2, 90161972CA2 90001862 34 90001862CD1 87 90001862CB190013122CA2 7503046 35 7503046CD1 88 7503046CB1 7503211 36 7503211CD1 897503211CB1 7503264 37 7503264CD1 90 7503264CB1 2515841CA2 90120235 3890120235CD1 91 90120235CB1 90120135CA2, 90141723CA2, 90141731CA290014961 39 90014961CD1 92 90014961CB1 7503199 40 7503199CD1 937503199CB1 7511530 41 7511530CD1 94 7511530CB1 7511535 42 7511535CD1 957511535CB1 7511536 43 7511536CD1 96 7511536CB1 7511583 44 7511583CD1 977511583CB1 7511395 45 7511395CD1 98 7511395CB1 90130146CA2 7511647 467511647CD1 99 7511647CB1 7510335 47 7510335CD1 100 7510335CB190057788CA2, 90057941CA2, 90078607CA2 7510337 48 7510337CD1 1017510337CB1 7510353 49 7510353CD1 102 7510353CB1 7510470 50 7510470CD1103 7510470CB1 7504648 51 7504648CD1 104 7504648CB1 7512747 527512747CD1 105 7512747CB1 7510146 53 7510146CD1 106 7510146CB1

TABLE 2 Polypep- Incyte GenBank ID NO: Proba- tide SEQ Polypep- orPROTEOME bility ID NO: tide ID ID NO: Score Annotation 1 7499940CD1g3293241 8.4E−135 [Homo sapiens] cyclic AMP-specific phosphodiesteraseHSPDE4A1A (Sullivan, M. et al. (1998) Biochem. J. 333 (Pt 3), 693-703) 23329870CD1 g5726647 6.9E−85 [Mus musculus] thioredoxin interactingfactor (Junn, E. et al. (2000) J. Immunol. 164 (12), 6287-6295) 37500698CD1 g11545707 3.1E−73 [Homo sapiens] ISCU2 (Tong, W. H. et al.(2000) EMBO J. 19 (21), 5692-5700) 4 7500223CD1 g3694659   1E−179 [Homosapiens] paraoxonase/arylesterase (Sulston, J. E. et al. (1998) GenomeRes. 8 (11), 1097-1108) 4 7500223CD1 337086|PON2   8E−180 [Homo sapiens][Hydrolase] Paraoxonase/arylesterase, member of a family that hydrolyzestoxic organophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification; variants altersusceptibility to parathion poisoning 4 7500223CD1 337084|PON1 9.5E−122[Homo sapiens] [Hydrolase] Paraoxonase (arylesterase), hydrolyzes toxicorganophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification; variants may affect theanti-atherosclerotic and anti- inflammatory response 4 7500223CD1326742|Pon1   2E−119 [Mus musculus][Hydrolase] Paraoxonase (A-esterase,aromatic esterase, arylesterase), member of a family that hydrolyzestoxic organophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification, may play a role inatherogenesis 5 7500295CD1 g3694659   1E−179 [Homo sapiens]paraoxonase/arylesterase (Sulston, J. E. et al. (1998) Genome Res. 8(11), 1097-1108) 5 7500295CD1 337086|PON2   8E−180 [Homo sapiens][Hydrolase] Paraoxonase/arylesterase, member of a family that hydrolyzestoxic organophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification; variants altersusceptibility to parathion poisoning 5 7500295CD1 337084|PON1 9.5E−122[Homo sapiens] [Hydrolase] Paraoxonase (arylesterase), hydrolyzes toxicorganophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification; variants may affect theanti-atherosclerotic and anti- inflammatory response 5 7500295CD1326742|Pon1   2E−119 [Mus musculus] [Hydrolase] Paraoxonase (A-esterase,aromatic esterase, arylesterase), member of a family that hydrolyzestoxic organophosphates, possibly functions in protecting low densitylipoprotein against oxidative modification, may play a role inatherogenesis 5 7502095CD1 729797|1fc4_A 1.1E−104 [Protein Data Bank]2-Amino-3-Ketobutyrate Coenzyme A Ligase 6 7502095CD1 g3342906 3.9E−217[Homo sapiens] 2-amino-3-ketobutyrate-CoA ligase (Edgar, A. J. et al.(2000) Eur. J. Biochem. 267: 1805-1812) 6 7502095CD1 729797|1fc4_A1.1E−104 [Protein Data Bank] 2-Amino-3-Ketobutyrate Coenzyme A Ligase 67502095CD1 251191.1|T25B9.1 4.1E−73 [Caenorhabditis elegans][Transferase] Member of the serine palmitoyltransferase protein family 77500507CD1 g3220249 9.6E−246 [Homo sapiens] 5-aminolevulinate synthase 2(Surinya, K. H. et al. (1998) J. Biol. Chem. 273: 16798-16809) 77500507CD1 665827|Alas2 4.8E−281 [Mus musculus][Transferase]5-aminolevulinic acid synthase, has strong similarity to human ALAS2,which catalyses the first step in heme biosynthesis; mutations in thehuman gene cause congenital sideroblastic anemia 7 7500507CD1339080|ALAS2 3.7E−246 [Homo sapiens][Transferase] Erythroid-specificdelta-aminolevulinate synthase, first step in heme biosynthesis;mutations in the gene cause congenital sideroblastic anaemia 77500507CD1 334122|ALAS1 9.6E−192 [Homo sapiens][Transferase]Delta-aminolevulinate synthase, catalyzes the first step in hemebiosynthesis 8 7500840CD1 g1220285 5.6E−15 [Schizosaccharomyces pombe]electron transfer protein 8 7500840CD1 371927|etp1   5E−16[Schizosaccharomyces pombe] Putative electron transfer protein, has highsimilarity to S. cerevisiae Cox15p 8 7500840CD1 644198|orf6.7220 1.2E−13[Candida albicans][Oxidoreductase] Member of the ferredoxin family ofelectron transport proteins that contain a2FE−2S cluster, has highsimilarity to uncharacterized S. cerevisiae Yah1p 8 7500840CD1340544|FDX1 1.7E−12 [Homo sapiens][Oxidoreductase; Smallmolecule-binding protein] [Cytoplasmic; Mitochondrial] Ferredoxin(adrenodoxin), an iron-sulfur protein that transfers electrons fromadrenodoxinreductase to P450scc, which is involved in steroid, vitaminD, and bile acid metabolism 9 7493620CD1 g516150 1.2E−249 [Homo sapiens]UDP-glucuronosyltransferase (Jin, C. J. et al. (1993) Biochem. Biophys.Res. Commun. 194: 496-503) 9 7493620CD1 338816| UGT2B7 7.2E−227 [Homosapiens] [Transferase][Endoplasmic reticulum; Cytoplasmic] Member of theUDP-glucuronosyltransferase 2B subfamily of endoplasmic reticulumglycoproteins that conjugate lipophilic aglycon substrates withglucuronic acid, glucuronidates 3,4-catechol estrogens and estriol 97493620CD1 344906| UGT2B11 2.2E−225 [Homo sapiens][Transferase][Endoplasmic reticulum; Cytoplasmic] Member of theUDP-glucuronosyltransferase 2B subfamily of endoplasmic reticulumglycoproteins that conjugate lipophilic aglycon substrates withglucuronic acid, possible substrates include polyhydroxylated estrogensand xenobiotics 9 7493620CD1 348401| UGT2B4   4E−217 [Homo sapiens][Transferase][Endoplasmic reticulum; Cytoplasmic]Bile acid UDPglycosyltransferase, member of the UDP-glucuronosyltransferase 2Bsubfamily of endoplasmic reticulum glycoproteins that conjugatelipophilic aglycon substrates with glucuronic acid 9 7493620CD1 338812|UGT2B15 2.9E−207 [Homo sapiens] [Transferase][Endoplasmic reticulum;Cytoplasmic] Member of the UDP-glucuronosyltransferase 2B subfamily ofendoplasmic reticulum glycoproteins that conjugate lipophilic aglyconsubstrates with glucuronic acid, glucuronidates several xenobiotics andsteroids 10 7494697CD1 g1088448 1.1E−155 [Homo sapiens] NADP dependentleukotriene b4 12-hydroxydehydrogenase (Yokomizo, T. et al. (1996) J.Biol. Chem. 271: 2844-2850) 10 7494697CD1 424790|   1E−156 [Homosapiens][Oxidoreductase] Leukotriene B4 12-hydroxydehydrogenase, LTB4DHconverts leukotriene B4 into the 12-oxo-derivative, inactivatingleukotriene B4 in non-leukocytes 10 7494697CD1 638338| 3.5E−28 [Candidaalbicans][Oxidoreductase] Member of the zinc-containing alcoholorf6.4290 dehydrogenase family, has low similarity to human LTB4DH,which is a leukotriene B4 12-hydroxydehydrogenase that convertsleukotriene B4 into the 12-oxo- derivative 11 8146738CD1 g125972936.9E−220 [Homo sapiens] acidic mammalian chitinase precursor (Boot, R.G. et al. (2001) J. Biol. Chem. 276: 6770-6778) 11 8146738CD1623690|TSA1902 1.8E−168 [Homo sapiens][Hydrolase] Protein with highsimilarity to chitotriosidase (CHIT1), a chitinase that is secreted byactivated macrophages and may function to degrade pathogen walls, memberof the glycosyl hydrolase 18 family 11 8146738CD1 712501|Ecf-1 2.2E−145[Mus musculus] Eosinophil chemotactic cytokine, a chitinase familyprotein chemotactic for eosinophils, bone marrow polymorphonuclearleukocytes, and T lymphocytes 11 8146738CD1 334648|CHIT1 1.5E−116 [Homosapiens] [Hydrolase][Extracellular (excluding cell wall)]Chitotriosidase (methylumbelliferyl tetra-N-acetyl-chitotetraosidehydrolase), a chitinase that is secreted by activated macrophages andmay function to degrade pathogen walls, mutations in the correspondinggene cause chitotriosidase deficiency 12 7500114CD1 g14714839 3.3E−129[Homo sapiens] 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase(hydroxymethylglutaricaciduria) 12 7500114CD1 347256|HMGCL 1.5E−120[Homo sapiens] [Lyase][Mitochondrial matrix; Cytoplasmic; Mitochondrial]3- Hydroxy-3-methylglutaryl Coenzyme A lyase, cleaves3-hydroxy-3-methylglutary CoA to acetoacetic acid and acetyl CoA, laststep of ketogenesis and leucine catabolism, functions in energymetabolism, deficiency leads to hypoglycemia and coma 13 7500197CD1g14603061 1.9E−202 [Homo sapiens] farnesyl diphosphate synthase(farnesyl pyrophosphate synthetase, dimethylallyltranstransferase,geranyltranstransferase) 13 7500197CD1 335298|FDPS 1.7E−203 [Homosapiens][Transferase] Farnesyl pyrophosphate synthetase(farnesyldiphosphate synthase), part of the cholesterol synthesis pathway 147500145CD1 g2121310 8.4E−176 [Homo sapiens] GP-39 cartilage protein (Rehli, M. et al. (1997) Genomics 43: 221-225.) 14 7500145CD1345056|CHI3L1 7.4E−177 [Homo sapiens][Structural protein;Hydrolase][Extracellular matrix (cuticle and basement membrane);Extracellular (excluding cell wall)] Cartilage glycoprotein- 39, hassimilarity to chitinases, expressed in rheumatoid arthritis cartilageand synovial cells (Hakala, B. E. et al. (1993) Human cartilage gp-39, amajor secretory product of articular chondrocytes and synovial cells, isa mammalian member of a chitinase protein family. J Biol Chem 268:25803-25810; Kirkpatrick, R. B. et al. (1997) Induction and expressionof human cartilage glycoprotein 39 in rheumatoid inflammatory andperipheral blood monocyte-derived macrophages. Exp. Cell Res. 237:46-54.) 14 7500145CD1 321804|Chi3l1 5.5E−129 [Musmusculus][Hydrolase][Extracellular (excluding cell wall)] Glycoprotein39, expressed in neu- and ras- but not c-myc (Myc)- or int-2-initiatedmammary tumors, has similarity to glycosylhydrolases (Morrison, B. W.,and Leder, P. (1994) neu and ras initiate murine mammary tumors thatshare genetic markers generally absent in c-myc and int-2-initiatedtumors. Oncogene 9: 3417-3426; Hakala, B. E. et al. (1993) supra; Jin,H. M., et al. (1998) Genetic characterization of the murine Ym1 gene andidentification of a cluster of highly homologous genes. Genomics 54:316-322.) 15 7500874CD1 g2121310 1.5E−66 [Homo sapiens] GP-39 cartilageprotein ( Rehli, M. et al. (1997) Genomics 43: 221-225.) 15 7500874CD1428668|PRDX5 1.9E−84 [Homosapiens][Oxidoreductase][Cytoplasmic;Mitochondrial; Peroxisome] Antioxidant enzyme, a member of a subfamilyof AhpC/TSA peroxiredoxin antioxidants, has peroxidase and antioxidantactivity and possibly functions in oxidative and inflammatory processes(Knoops, B., et al. (1999) Cloning and characterization of AOEB166, anovel mammalian antioxidant enzyme of the peroxiredoxin family. J BiolChem 274: 30451-30458; Yamashita, H. et al. (1999) Characterization ofhuman and murine PMP20 peroxisomal proteins that exhibit antioxidantactivity in vitro. J Biol Chem 274: 29897-29904; Wattiez, R. et al.(1999) supra.) 15 7500874CD1 430156|Pmp20 1.5E−50 [Musmusculus][Oxidoreductase][Cytoplasmic; Peroxisome] Peroxiredoxin V, athioredoxin peroxidase that prevents p53 (Tp53)-dependent generation ofreactive oxygen species and inhibits p53-induced apoptosis, functions inredox signaling (Zhou, Y., et al. (2000) Mouse peroxiredoxin V is athioredoxin peroxidase that inhibits p53-induced apoptosis. Biochem.Biophys. Res. Commun. 268: 921-927). 16 7500495CD1 g6103724 2.2E−83[Homo sapiens] antioxidant enzyme B166 (Andresen, B. S. et al. (1996)Hum. Mol. Genet. 5: 461-472.) 16 7500495CD1 428668|PRDX5 1.9E−84[Homosapiens][Oxidoreductase][Cytoplasmic; Mitochondrial; Peroxisome]Antioxidant enzyme, a member of a subfamily of AhpC/TSA peroxiredoxinantioxidants, has peroxidase and antioxidant activity and possiblyfunctions in oxidative and inflammatory processes (Knoops, B., et al.(1999) Cloning and characterization of AOEB166, a novel mammalianantioxidant enzyme of the peroxiredoxin family. J Biol Chem 274:30451-30458; Yamashita, H. et al. (1999) Characterization of human andmurine PMP20 peroxisomal proteins that exhibit antioxidant activity invitro. J Biol Chem 274: 29897-29904; Wattiez, R. et al. (1999) supra.)16 7500495CD1 430156|Pmp20 1.5E−50 [Musmusculus][Oxidoreductase][Cytoplasmic; Peroxisome] Peroxiredoxin V, athioredoxin peroxidase that prevents p53 (Tp53)-dependent generation ofreactive oxygen species and inhibits p53-induced apoptosis, functions inredox signaling (Zhou, Y., et al. (2000) Mouse peroxiredoxin V is athioredoxin peroxidase that inhibits p53-induced apoptosis. Biochem.Biophys. Res. Commun. 268: 921-927). 17 7500194CD1 g790447 1.1E−175[Homo sapiens] very-long-chain acyl-CoA dehydrogenase (Andresen, B. S.et al. (1996) Hum. Mol. Genet 5: 461-472.) 17 7500194CD1 339036|ACADVL9.4E−177 [Homo sapiens][Oxidoreductase][Cytoplasmic; Mitochondrial] Verylong chain acyl-Coenzyme A dehydrogenase, oxidizes straight chainacyl-CoAs in the initial step of fatty acid beta-oxidation, deficiencydue to mutation in the gene causes sudden infant death syndrome andhypertrophic cardiomyopathy (Aoyama, T. et al. (1995) Cloning of humanvery-long-chain acyl-coenzyme A dehydrogenase and molecularcharacterization of its deficiency in two patients. Am. J. Hum. Genet.57: 273-283; Strauss, A. W. et al. (1995) Molecular basis of humanmitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causingcardiomyopathy and sudden death in childhood. Proc Natl Acad Sci USA 92:10496-10500.) 18 7500871CP1 g14919433 3.8E−164 [Homo sapiens] Similar tochitinase 3-like 1 (cartilage glycoprotein-39) 18 7500871CD1345056|CHI3L1 1.1E−164 [Homo sapiens][Structural protein;Hydrolase][Extracellular matrix (cuticle and basement membrane);Extracellular (excluding cell wall)] Cartilage glycoprotein- 39, hassimilarity to chitinases, expressed in rheumatoid arthritis cartilageand synovial cells (Hakala, B. E. et al. (1993) supra: Kirkpatrick, R.B. et al. (1997) supra.) 18 7500871CD1 321804|Chi3l1 4.5E−122 [Musmusculus][Hydrolase][Extracellular (excluding cell wall)] Glycoprotein39, expressed in neu- and ras- but not c-myc (Myc)- or int-2-initiatedmammary tumors, has similarity to glycosylhydrolases supra(Morrison, B.W., and Leder, P. (1994) supra: Hakala, B. E. et al. (1993) supra; Jin,H. M., et al. (1998) supra.) 19 7500873CD1 g14919433 4.6E−120 [Homosapiens] Similar to chitinase 3-like 1 (cartilage glycoprotein-39) 197500873CD1 345056|CHI3L1 1.4E−120 [Homo sapiens][Structural protein;Hydrolase][Extracellular matrix (cuticle and basement membrane);Extracellular (excluding cell wall)] Cartilage glycoprotein- 39, hassimilarity to chitinases, expressed in rheumatoid arthritis cartilageand synovial cells (Hakala, B. E. et al. (1993) supra; Kirkpatrick, R.B. et al. (1997) supra.) 19 7500873CD1 321804|Chi3l1 1.5E−89 [Musmusculus][Hydrolase][Extracellular (excluding cell wall)] Glycoprotein39, expressed in neu- and ras- but not c-myc (Myc)- or int-2-initiatedmammary tumors, has similarity to glycosylhydrolases (Morrison, B. W.,and Leder, P. (1994) supra; Hakala, B. E. et al. (1993) supra; Jin, H.M., et al. (1998) supra.) 20 7503491CD1 g4151819 1.8E−186 [Homo sapiens]uroporphyrinogen decarboxylase 20 7503491CD1 720887|1uro_A 1.5E−187[Protein Data Bank] Uroporphyrinogen Decarboxylase 20 7503491CD1606326|UROD 1.5E−187 [Homo sapiens] [Lyase] Uroporphyrinogendecarboxylase, catalyzes decarboxylation of the four acetyl side chainsof uroporphyrinogen III to form coproporphyrinogen III inhemebiosynthesis; deficiency causes familial porphyria cutanea tarda andhepatoerythropoietic porphyria Moran-Jimenez, M. J. et al. (1996) Am. J.Hum. Genet. 58: 712-721 Uroporphyrinogen decarboxylase: complete humangene sequence and molecular study of three families withhepatoerythropoietic porphyria. Am J Hum Genet 58, 712-21 (1996). 207503491CD1 326094|Urod 2.3E−171 [Mus musculus] [Lyase] Uroporphyrinogendecarboxylase, catalyzes decarboxylation of the four acetyl side chainsof uroporphyrinogen III to form coproporphyrinogen III in hemebiosynthesis 20 7503491CD1 367482|Urod 3.5E−166 [Rattus norvegicus][Lyase] Uroporphyrinogen decarboxylase, has strong similarity to humanUROD, which catalyzes decarboxylation of the four acetyl side chains ofuroporphyrinogen III to form coproporphyrinogen III in heme biosynthesis20 7503491CD1 646474|orf6.8358 2.7E−87 [Candida albicans] [Lyase]Protein with high similarity to S. cerevisiae Hem12p, which isuroporphyrinogen decarboxylase that carries out decarboxylation ofuroporphyrinogen acetyl side chains to yield coproporphyrinogen, memberof the uroporphyrinogen-decarboxylase (URO-D) family 21 7503427CD1g190818 1.2E−101 [Homo sapiens] quinone oxidoreductase (Jaiswal, A. K.,et al (1990) Biochemistry 29: 1899-1906) 21 7503427CD1 336626|NMOR21.1E−102 [Homo sapiens] [Oxidoreductase] NAD(P)H:quinoneoxidoreductase,flavoprotein that oxidizes NADH or NADPH byquinones andoxidation-reduction dyes 7503427CD1 727253|1qr2_A 3.6E−102 [Protein DataBank] Quinone Reductase Type 2 21 7503427CD1 611228|Nmor2 5.1E−82 [Musmusculus] [Oxidoreductase] NRH: quinone oxidoreductase, has strongsimilarity to human NMOR2, which is a flavoprotein that oxidizes NADH orNADPH by quinones and oxidation-reduction dyes 21 7503427CD1 336624|DIA47.5E−43 [Homo sapiens] [Oxidoreductase] [Cytoplasmic; Axon] Cytochromeb5reductase, reduces redox dyes and quinones and may protect againstcancer caused by quinones and their precursors; mutations in thecorresponding gene are associated with an increased risk of benzenehematotoxicity 21 7503427CD1 722688|1d4a_A 2.5E−42 [Protein Data Bank]Quinone Reductase 22 7503547CD1 g181553 1.6E−91 [Homo sapiens]dihydropteridine reductase (EC 1.6.99.7) (Lockyer, J. et al. (1987)Proc. Natl. Acad. Sci. U.S.A. 84: 3329-3333) 22 7503547CD1726758|1hdr_(—) 1.1E−92 [Protein Data Bank] Dihydropteridine Reductase(Dhpr) 22 7503547CD1 337462|QDPR 1.4E−92 [Homo sapiens] [Oxidoreductase]Dihydropteridine reductase, catalyzes the NADH-dependent reduction ofdihydrobiopterin, required for pterin-dependent hydroxylating systems ofaromatic amino acids 22 7503547CD1 718799|1dhr_(—) 4.1E−73 [Protein DataBank] Dihydropteridine Reductase (Dhpr) (E.C.1.6.99.7) 22 7503547CD1628635|Qdpr 4.1E−73 [Rattus norvegicus] [Oxidoreductase]Dihydropteridine reductase, has very strong similarity to human QDPR,which reduces quinonoid dihydrobiopterin and is required forpterin-dependent hydroxylating systems of aromatic amino acid 227503547CD1 249586|T03F6.1 1.2E−43 [Caenorhabditis elegans] Protein withstrong similarity to human quinoid dihydropteridine reductaseQDPR(Hs.75438) 23 1932641CD1 g4159682 2.4E−281 [Cricetulus griseus]Phosphatidylglycerophosphate synthase (Kawasaki, K. (1999) J. Biol.Chem. 274: 1828-1834) 23 1932641CD1 605378| 3.6E−145 [Homo sapiens]Protein of unknown function, has low similarity to a region of S.DKFZp762M186 cerevisiae Pgs1p, which is a phosphatidyl glycerophosphatesynthase 23 1932641CD1 715208|PGS1 4.5E−60 [Saccharomyces cerevisiae][Transferase] [Endoplasmic reticulum; Plasma membrane; Mitochondrialouter membrane; Mitochondrial] Phosphatidyl glycerophosphate synthase,the first enzyme of the cardiolipin biosynthetic pathway 23 1932641CD1646720|orf6.8481 4.6E−58 [Candida albicans] [Transferase] Protein withhigh similarity to S. cerevisiae Pgs1p, which is a phosphatidylglycerophosphate synthase and the first enzyme of the cardiolipinbiosynthetic pathway, member of the phospholipaseD/transphosphatidylasefamily 23 1932641CD1 657982| 1.4E−38 [Schizosaccharomyces pombe]Putative phosphatidylglycerophosphate synthase, SPBP18G5.02 the firstenzyme of the cardiolipin biosynthetic pathway 24 6892447CD1 g124841496.1E−62 [Cochliobolus heterostrophus] peptide synthetase-like protein 246892447CD1 424014|KIAA0934   0.0 [Homo sapiens] Protein containing anAMP-binding domain 24 6892447CD1 424244|KIAA0184   0.0 [Homo sapiens]Protein containing an AMP-binding domain 25 7503416CD1 g12655193   0.0[Homo sapiens] phosphoenolpyruvate carboxykinase 2 (mitochondrial) 257503416CD1 341026|PCK2   0.0 [Homo sapiens] [Lyase; Other kinase][Cytoplasmic; Mitochondrial] Phosphoenolpyruvate carboxykinase,catalyzes the formation of phosphoenolpyruvate by decarboxylation ofoxaloacetate, rate-limiting step of gluconeogenesis 25 7503416CD1368648|Pck1   2E−240 [Mus musculus] [Lyase; Other kinase] [Cytoplasmic]Phosphoenolpyruvate carboxykinase, catalyzes the formation ofphosphoenolpyruvate by decarboxylation of oxaloacetate 25 7503416CD1336802|PCK1   7E−238 [Homo sapiens] [Lyase; Other kinase] [Cytoplasmic]Cyto Solic phosphoenolpyruvate carboxykinase (GTP)(GTP:oxaloacetatecarboxy-lyase (transphosphorylating)), catalyzes theformation of phosphoenolpyruvate by decarboxylation of oxaloacetate,rate-limiting step of gluconeogenesis Rucktaschel, A. K. et al. (2000)Biochem. J. 352: 211-217 Regulation by glucagon (cAMP) and insulin ofthe promoter of the human phosphoenolpyruvate carboxykinase gene(cytosolic) in cultured rat hepatocytes and in human hepatoblastomacells 25 7503416CD1 249071|R11A5.4 2.9E−195 [Caenorhabditis elegans][Lyase] [Mitochondrial matrix; Mitochondrial] Member of thephosphoenolpyruvate carboxykinase protein family 25 7503416CD1251847|W05G11.6 6.5E−189 [Caenorhabditis elegans] [Lyase] [Mitochondrialmatrix; Mitochondrial] Member of the phosphoenolpyruvate carboxykinaseprotein family 26 7503874CD1 g3335098 7.6E−241 [Homo sapiens] CD39L2(Chadwick, B. P. and Frischauf, A. M. (1998) Genomics 50: 357-367) 267503874CD1 339194|ENTPD6 6.7E−242 [Homo sapiens] [Hydrolase; ATPase]Member of the CD39-like family, a putative ecto-apyrase 26 7503874CD1339198|ENTPD5 4.2E−97 [Homo sapiens] [Hydrolase; ATPase] Member of theCD39-like family, a putative ecto-apyrase 26 7503874CD1 583749|Entpd53.5E−87 [Mus musculus] [Other phosphatase; Hydrolase] [Endoplasmicreticulum; Cytoplasmic] Endoplasmic reticulum nucleoside diphosphatase,hydrolyzes UDP to UMP, which may promote reglucosylation reactionsinvolved in glycoprotein folding and quality control in the endoplasmicreticulum, member of the CD39- like family 27 7503454CD1 g123142362.9E−115 [Homo sapiens] bA127L20.1 (novel glutathione-S-transferase) 277503454CD1 340658|GSTTLp28 7.5E−79 [Homo sapiens] [Transferase] Memberof a family of GSTomega class proteins that have glutathione-dependentthioltransferase activity and glutathione- dependent dehydroascorbatereductase activity Board, P. G. et al. (2000) J. Biol. Chem. 275:24798-24806 Identification, characterization, and crystal structure ofthe omega class glutathione transferases. 27 7503454CD1 718283|1eem_A7.5E−79 [Protein Data Bank] Glutathione-S-Transferase 27 7503454CD1429880|Gsttl 4.2E−68 [Mus musculus] [Small molecule-binding protein][Nuclear; Cytoplasmic] Member of a family of GST-like proteins that bindglutathione but have no apparent transferase or peroxidase activity 277503454CD1 248040|K10F12.4 2.2E−28 [Caenorhabditis elegans][Transferase] [Cytoplasmic] Member of the glutathione S-transferaseprotein family, has similarity to human and S. cerevisiae glutathioneS-transferases 27 7503454CD1 242759|F13A7.10 8.6E−25 [Caenorhabditiselegans] [Transferase] [Cytoplasmic] Member of the glutathioneS-transferase protein family, has similarity to human and S. cerevisiaeglutathione S-transferases 28 7503528CD1 g12654777 1.6E−110 [Homosapiens] glutathione S-transferase subunit 13 homolog 29 7503705CD1g1504040 7.8E−89 [Homo sapiens] (D86983) similar to D. melanogasterperoxidasin(U11052) (Nagase, T. et al. (1996) DNA Res. 3: 321-329.) 297503705CD1 628843|D2S448 6.8E−90 [Homo sapiens] Peroxidasin (melanomaassociated), has similarity to Drosophila peroxidasin, which is anextracellular matrix-associated peroxidase (Horikoshi, N. et al. (1999)Isolation of differentially expressed cDNAs from p53- dependentapoptotic cells: activation of the human homologue of the Drosophilaperoxidasin gene. Biochem. Biophys. Res. Commun. 261: 864-869.) 297503705CD1 344170|EPX   4E−27 [Homo sapiens][Oxidoreductase] Eosinophilperoxidase, participates in host defense against extracellular pathogensthrough the generation of reactive oxidants; may play a role in tissuedamage in asthma and other chronic inflammatory conditions (Henderson,J. P. et al. (2001) Bromination of deoxycytidine by eosinophilperoxidase: a mechanism for mutagenesis by oxidative damage ofnucleotide precursors. Proc. Natl. Acad. Sci. USA 98: 1631-1636.) 307503707CD1 g1504040   0.0 [Homo sapiens] (D86983) similar to D.melanogaster peroxidasin(U11052) (Nagase, T. et al. (1996) DNA Res. 3:321-329.) 30 7503707CD1 628843|D2S448   0.0 [Homo sapiens] Peroxidasin(melanoma associated), has similarity to Drosophila peroxidasin, whichis an extracellular matrix-associated peroxidase (Horikoshi, N. et al.(1999) supra.) 30 7503707CD1 429244|Tpo 1.4E−129 [Musmusculus][Oxidoreductase] Thyroid peroxidase, required for synthesis ofthyroid hormones; expression of the rat homolog Rn.9957 is induced byTSH (Kotani, T. et al. (1993) Nucleotide sequence of the cDNA encodingmouse thyroid peroxidase. Gene 123: 289-290; Nguyen, L. Q. et al. (2000)A dominant negative CREB (cAMP response element-binding protein) isoforminhibits thyrocyte growth, thyroid-specific gene expression,differentiation, and function. Mol. Endocrinol. 14: 1448-1461.) 3190001962CD1 g7533024 1.4E−189 [Homo sapiens] oxysterol7alpha-hydroxylase (Li-Hawkins, J. et al. (2000) J. Biol. Chem. 275:16543-16549.) 31 90001962CD1 476053|CYP39A1 1.3E−190 [Homosapiens][Oxidoreductase; Small molecule-binding protein][Endoplasmicreticulum; Cytoplasmic; Microsomal fraction] Oxysterol 7alpha-hydroxylase, a microsomal cytochrome P450 enzyme that convertsoxysterols to 7 alpha- hydroxylated bile acids, prefers24-hydroxycholesterol, expressed in liver (Li-Hawkins, J. et al. (2000)supra.) 31 90001962CD1 340310|CYP7B1 8.7E−39 [Homosapiens][Oxidoreductase; Small molecule-binding protein][Endoplasmicreticulum; Microsomal fraction; Cytoplasmic] Oxysterol7alpha-hydroxylase, a cytochrome P450 enzyme, functions in the acidicpathway of bile acid biosynthesis; mutations in the corresponding genecause severe neonatal cholestatic liver disease (Setchell, K. D. et al.(1998) Identification of a new inborn error in bile acid synthesis:mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatalliver disease. J. Clin. Invest. 102: 1690-1703). 31 90001962CD1583943|Cyp7b1 5.2E−38 [Mus musculus][Oxidoreductase; Transporter; Smallmolecule-binding protein] Cytochrome P450 that possibly functions inbrain steroid metabolism, expressed primarily in brain (Stapleton, G. etal. (1995) A novel cytochrome P450 expressed primarily in brain. J.Biol. Chem. 270: 29739-29745). 32 70819231CD1 g4760647 4.5E−190 [Homosapiens] phospholipase (Tani, K. et al. (1999) p125 is a novel mammalianSec23p-interacting protein with structural similarity tophospholipid-modifying proteins. J. Biol. Chem. 274: 20505-20512.) 3270819231CD1 423709|KIAA0725   0.0 [Homo sapiens] Protein which has highsimilarity to a region of human P125, which is Sec23-interactingprotein, has similarity to phosphatidic acid preferring- phospholipaseA1, may act in the early secretory pathway 32 70819231CD1 428430|P1253.9E−191 [Homo sapiens][Small molecule-binding protein][Golgi;Endoplasmic reticulum; Cytoplasmic] Sec23-interacting protein, hassimilarity to phosphatidic acid preferring-phospholipase A1, binds tothe COPII vesicle coat protein Sec23p, and may play a role in the earlysecretory pathway (Tani, K. et al. (1999) supra; Mizoguchi, T. et al.(2000) Determination of functional regions of p125, a novel mammalianSec23p-interacting protein. Biochem. Biophys. Res. Commun. 279:144-149.) 33 7504066CD1 g189246 1.3E−71 [Homo sapiens] NAD(P)H:menadioneoxidoreductase (Jaiswal, A. K. et al. (1988) J. Biol. Chem. 263:13572-13578.) 33 7504066CD1 331838|Rn.11234 1.7E−108 [Rattusnorvegicus][Oxidoreductase][Cytoplasmic] Quinone reductase(NAD(P)H:menadione oxidoreductase), cytosolic reductase targetingquinones which functions in stress responses; human DIA4 deficiency isassociated with increased benzene hematotoxicity, urolithiasis andvarious cancers (Jaiswal, A. K. (1991) Human NAD(P)H:quinoneoxidoreductase (NQO1) gene structure and induction by dioxin.Biochemistry 30: 10647-10653; Yonehara, N. et al. (1997) Involvement ofnitric oxide in re-innvervation of rat molar tooth pulp followingtransaction of the inferior alveolar nerve. Brain Res. 757: 31-36.) 3490001862CD1 g2443331 3.1E−258 [Xenopus laevis] Nfrl (Hatada, S. et al.(1997) Gene 194 (2), 297-299) 34 90001862CD1 715427|F20D6.11 5.2E−82[Caenorhabditis elegans][Oxidoreductase] Putative oxidoreductase, hasweak similarity to human and S. cerevisiae dihydrolipoamidedehydrogenases 34 90001862CD1 372246| 1.5E−28 [Schizosaccharomycespombe] Putative flavoprotein SPAC29A4.01c 34 90001862CD1 718217|1d7y_A5.4E−28 [Protein Data Bank] Ferredoxin Reductase 34 90001862CD1339966|PDCD8 1.3E−23 [Homo sapiens][Oxidoreductase; Smallmolecule-binding protein][Nuclear, Cytoplasmic; Mitochondrial]Programmed cell death 8 (apoptosis-inducing factor), acaspase-independent apoptotic protease activator and flavoprotein,translocates from the mitochondria to the nucleus to play a role inchromatin condensation and DNA fragmentation 34 90001862CD1 704471|Pdcd81.6E−22 [Rattus norvegicus] Programmed cell death 8 (apoptosis-inducingfactor), an apoptosis activator that translocates from the mitochondriato the nucleus to play a role in DNA fragmentation during inducedphotoreceptor apoptosis 35 7503046CD1 g1854550 1.4E−230 [Mus musculus]red-1 (Kurooka, H. et al. (1997) Genomics 39 (3), 331-339) 35 7503046CD1326490|Nxn 1.2E−231 [Mus musculus][Oxidoreductase][Nuclear] Putativenucleoredoxin, may modify cysteine residues in DNA-binding domains oftranscription factors 36 7503211CD1 g181333   6E−232 [Homo sapiens]steroid 11-beta-hydroxylase (Mornet, E. et al. (1989) J. Biol. Chem. 264(35), 20961-20967) 36 7503211CD1 709557|CYP11B1 5.2E−233 [Homosapiens][Oxidoreductase; Small molecule-binding protein][Cytoplasmic;Mitochondrial] Steroid 11 beta-hydroxylase, a cytochrome P450 thatconverts 11 deoxycortisol to cortisol; deficiency causes hypertensivecongenital adrenal hyperplasia, and fusion of the gene with other genesis associated with diseases of aldosterone synthesis 36 7503211CD1709559|CYP11B2 5.5E−216 [Homo sapiens][Oxidoreductase; Transporter;Small molecule-binding protein] Cytochrome P450 subfamily XIBpolypeptide 2, synthesizes aldosterone; mutations in the correspondinggene cause hyperaldosteronism, aldosterone synthase deficiency type I,corticosterone methyloxidase I deficiency, and cardiac hypertrophy 367503211CD1 697979|Cyp11b2 1.6E−157 [Rattus norvegicus][Oxidoreductase]Aldosterone synthase, a cytochrome P450 11 betahydroxylase/aldosterone-2 synthase, converts 11-deoxycorticosterone toaldosterone, corticosterone, and 18-hydroxy corticosterone 36 7503211CD1422985|Cyp11b1 3.9E−156 [Rattus norvegicus][Oxidoreductase; Transporter;Small molecule-binding protein] P450 11-beta hydroxylase, acts inmineral corticoid and glucocorticoid biosynthesis within the adrenal toconvert 11-deoxycoiticosterone to corticosterone and 18hydroxydeoxycorticosterone 36 7503211CD1 590009|Cyp11b 4.2E−146 [Rattusnorvegicus][Oxidoreductase; Transporter; Small molecule-binding protein]Cytochrome P450 11beta, acts in mineralocorticoid biosynthesis toconvert 11 deoxycorticosterone to corticosterone and 18 hydroxy 11deoxycorticosterone, may help regulate blood pressure 37 7503264CD1g4960208 9.5E−151 [Homo sapiens] inorganic pyrophosphatase (Fairchild,T. A. et al. (1999) Biochim. Biophys. Acta 1447 (2-3), 133-136) 377503264CD1 622055|PP 8.3E−152 [Homo sapiens][Other phosphatase;Hydrolase] Inorganic pyrophosphatase, catalyzes the hydrolysis ofpyrophosphate to inorganic phosphate (Pi) 37 7503264CD1 439569|C47E12.46.7E−76 [Caenorhabditis elegans][Otherphosphatase;Hydrolase][Cytoplasmic] Member of the inorganic pyrophosphatase proteinfamily 37 7503264CD1 697512|SID6-306 6.0E−75 [Homo sapiens] Protein withhigh similarity to inorganic pyrophosphatase (PP) 37 7503264CD15980|IPP1 7.6E−75 [Saccharomyces cerevisiae][Otherphosphatase;Hydrolase][Cytoplasmic] Inorganic pyrophosphatase, cytoplasmic 377503264CD1 717086|1e6a_A 1.2E−74 [Protein Data Bank] InorganicPyrophosphatase 38 90120235CD1 g2408127 7.9E−19 [Trypanosoma cruzi]glycosylphosphatidylinositol-specific phospholipase C (Redpath, M. B. etal. (1998) Mol. Biochem. Parasitol. 94 (1), 113-121) 39 90014961CD1g2634852 2.4E−20 [Bacillus subtilis] similar to glycerophosphodiesterphosphodiesterase (Kunst, F. et al. (1997) Nature 390 (6657), 249-256)39 90014961CD1 370061| 2.7E−13 [Schizosaccharomyces pombe] Protein withweak similarity to glycerophosphoryl SPAC4D7.02c diesterphosphodiesterases 40 7503199CD1 g3293241 5.9E−81 [Homo sapiens] cyclicAMP-specific phosphodiesterase HSPDE4A1A (Sullivan, M. et al. (1998)Biochem. J. 333: 693-703.) 40 7503199CD1 344690|PDE4A 5.2E−82 [Homosapiens][Hydrolase][Plasma membrane] cAMP-specific phosphodiesterasethat is sensitive to the antidepressant rolipram, has similarity toDrosophila dnc, which is the affected protein in the learning and memorymutant dunce (Huston, E. et al. (1996) J. Biol. Chem. 271: 31334-31344.)40 7503199CD1 329794|Pde4a 9.8E−71 [Rattusnorvegicus][Hydrolase][Cytoplasmic] cAMP-specific phosphodiesterase thatis sensitive to the antidepressant rolipram, has similarity toDrosophila dnc, the affected protein in the learning and memory mutantdunce (Davis, R. L. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3604-3608.) 41 7511530CD1 g4151815 6.5E−21 [Homo sapiens]uroporphyrinogen decarboxylase 41 7511530CD1 606326|UROD 5.2E−22 [Homosapiens][Lyase] Uroporphyrinogen decarboxylase, catalyzes conversion ofuroporphyrinogen I or III to coproporphyrinogen I or III in the hemebiosynthetic pathway; mutations in the UROD gene cause familialporphyria cutanea tarda and hepatoerythropoietic porphyria 41 7511530CD1Phillips, J. D. et al., A mouse model of familial porphyria cutaneatarda., Proc Nati Acad Sci USA 98, 259-264. (2001). 41 7511530CD1McManus, J. F. et al.. Five new mutations in the uroporphyrinogendecarboxylase gene identified in families with cutaneous porphyria.,Blood 88, 3589-600. (1996). 42 7511535CD1 g4151815 4.2E−136 [Homosapiens] uroporphyrinogen decarboxylase 42 7511535CD1 606326|UROD3.4E−137 [Homo sapiens][Lyase] Uroporphyrinogen decarboxylase, catalyzesconversion of uroporphyrinogen I or III to coproporphyrinogen I or IIIin the heme biosynthetic pathway; mutations in the UROD gene causefamilial porphyria cutanea tarda and hepatoerythropoietic porphyria 427511535CD1 Phillips, J. D. et al. (supra) 42 7511535CD1 McManus, J. F.et al. (supra) 43 7511536CD1 g2905794 9.2E−169 [Homo sapiens]uroporphyrinogen decarboxylase 43 7511536CD1 606326|UROD 8.4E−169 [Homosapiens][Lyase] Uroporphyrinogen decarboxylase, catalyzes conversion ofuroporphyrinogen I or III to coproporphyrinogen I or III in the hemebiosynthetic pathway; mutations in the UROD gene cause familialporphyria cutanea tarda and hepatoerythropoietic porphyria 43 7511536CD1Phillips, J. D. et al. (supra) 43 7511536CD1 McManus, J. F. et al.(supra) 44 7511583CD1 g12653601 7.7E−73 [Homo sapiens] quinoiddihydropteridine reductase 44 7511583CD1 337462|QDPR 1.3E−73 [Homosapiens][Oxidoreductase] Quinoid dihydropteridine reductase, catalyzesthe NADH-dependent reduction of dihydrobiopterin, required for pterin-dependent hydroxylating systems of aromatic amino acids; mutations inthe corresponding gene cause atypical phenylketonuria 44 7511583CD1Sumi-Ichinose, C. et al., Catecholamines and Serotonin Are DifferentlyRegulated by Tetrahydrobiopterin. A STUDY FROM 6-PYRUVOYLTETRAHYDROPTERIN SYNTHASE KNOCKOUT MICE., J Biol Chem 276,41150-60. (2001). 44 7511583CD1 628635|Qdpr 5.8E−71 [Rattusnorvegicus][Oxidoreductase] Quinoid dihydropteridine reductase,catalyzes the NADH-dependent reduction of dihydrobiopterin; mutations inhuman QDPR cause atypical phenylketonuria 44 7511583CD1 Pereon, Y. etal., Chronic stimulation differentially modulates expression of mRNA fordihydropyridine receptor isoforms in rat fast twitch skeletal muscle.,Biochem Biophys Res Commun 235, 217-22 (1997). 45 7511395CD1 g5161506.1E−242 [Homo sapiens] UDP-glucuronosyltransferase (Jin, C. J. et al.,(1993) Biochem. Biophys. Res. Commun. 194, 496-503) 45 7511395CD1338810|UGT2B10 4.9E−243 [Homo sapiens][Transferase][Endoplasmicreticulum; Cytoplasmic] UDP glycosyltransferase 2 polypeptide B10, aUDP-glucuronosyltransferase for which no substrate has been found,likely to play a role in glucuronidation which inactivates and increasesthe polarity of substrates and allows them to be more easily excreted 457511395CD1 Turgeon, D. et al., Relative Enzymatic Activity, ProteinStability, and Tissue Distribution of Human Steroid-Metabolizing UGT2BSubfamily Members., Endocrinology 142, 778-787. (2001). 45 7511395CD1344906|UGT2B11 1.4E−223 [Homo sapiens][Transferase][Endoplasmicreticulum; Cytoplasmic] UDP glycosyltransferase 2 polypeptide B11, aUDP-glucuronosyltransferase for which no substrate has been found,likely to play a role in glucuronidation which inactivates and increasesthe polarity of substrates and allows them to be more easily excreted 457511395CD1 Strassburg, C. P. et al. Polymorphic Gene Regulation andInterindividual Variation of UDP-glucuronosyltransferase Activity inHuman Small Intestine., J Biol Chem 275, 36164-36171 (2000). 467511647CD1 g4808241 3.4E−31 [Homo sapiens] dJ466N1.2 (glycineC-acetyltransferase (2-amino-3-ketobutyrate coenzyme A ligase)) 467511647CD1 569126|GCAT 2.7E−32 [Homo sapiens] Protein containing twoaminotransferase class I and II domains, which are found in somepyridoxal-dependent enzymes, has low similarity to serinepalmitoyltransferase long chain base subunit 1 (human SPTLC1), which isinvolved in ceramide biosynthesis 46 7511647CD1 587005|Gcat 2.8E−19 [Musmusculus] Protein of unknown function, has moderate similarity to aregion of erythroid-specific delta-aminolevulinate synthase (humanALAS2), which catalyzes the first step in heme biosynthesis 477510335CD1 g12653261 5.7E−130 [Homo sapiens] acyl-Coenzyme Adehydrogenase, very long chain 339036|ACADVL 4.6E−131 [Homosapiens][Oxidoreductase][Cytoplasmic; Mitochondrial] Very long chainacyl-Coenzyme A dehydrogenase, oxidizes straight chain acyl-CoAs in theinitial step of fatty acid beta-oxidation, deficiency due to mutation inthe gene causes sudden infant death syndrome and hypertrophiccardiomyopathy. Aoyama, T. et al. (1995) Am J Hum Genet 57: 273-283.589769|Acadvl 1.4E−104 [Rattus norvegicus][Oxidoreductase][Cytoplasmic;Mitochondrial] Very-long- chain acyl-CoA dehydrogenase, rate-controllingenzyme in beta-oxidation of long- chain fatty acids. Aoyama, T. et al.(1994) J Biol Chem 269: 19088-19094. 48 7510337CD1 g12653261   0.0[fl][Homo sapiens] acyl-Coenzyme A dehydrogenase, very long chain339036|ACADVL   0 [Homo sapiens][Oxidoreductase][Cytoplasmic;Mitochondrial] Very long chain acyl-Coenzyme A dehydrogenase, oxidizesstraight chain acyl-CoAs in the initial step of fatty acidbeta-oxidation, deficiency due to mutation in the gene causes suddeninfant death syndrome and hypertrophic cardiomyopathy. Aoyama, T. et al.(1995) Am. J. Hum. Genet. 5: 273-283. 608019|Acadvl 1.8E−278 [Musmusculus][Oxidoreductase][Cytoplasmic; Mitochondrial] Very-long-chainacyl coenzyme A dehydrogenase, involved in beta-oxidation of long-chainfatty acids. She, P. et al. (2000) Mol. Cell. Biol. 20: 6508-6517. 497510353CD1 g14603061 4.8E−227 [Homo sapiens] farnesyl diphosphatesynthase (faraesyl pyrophosphate synthetase,dimethylallyltranstransferase, geranyltranstransferase) 50 7510470CD1g181333   4E−200 [Homo sapiens] steroid 11-beta-hydroxylase 517504648CD1 g790447 4.2E−253 [Homo sapiens] very-long-chain acyl-CoAdehydrogenase (Andresen, B. S. et al. (1996) Hum. Mol. Genet. 5,461-472) 51 7504648CD1 339036|ACADVL 3.5E−254 [Homosapiens][Oxidoreductase][Cytoplasmic;Mitochondrial] Very long chainacyl-coenzyme A dehydrogenase, oxidizes straight chain acyl-CoAs in theinitial step of fatty acid beta-oxidation, severe deficiency results ininfant cardiomyopathy with high mortality, mild deficiency results inhypoketotic hypoglycemia. Aoyama, T. et al. Am J Hum Genet 57, 273-83(1995); Aoyama, T. et al. Biochem Biophys Res Commun 191, 1369-72(1993); Strauss, A. W. et al. Proc Natl Acad Sci USA 92, 10496-500(1995); Bonnet, D. et al. Circulation 100, 2248-53. (1999); Andresen, B.S. et al. Am J Hum Genet 64, 479-94. (1999). 52 7512747CD1 g44546903.1E−95 [Homo sapiens] glutathione S-transferase subunit 13 homolog(Zhang, Q. H. et al., (2000) Genome Res. 10, 1546-1560) 52 7512747CD1475637|LOC51064 2.4E−96 [Homo sapiens] Member of the2-hydroxychromene-2-carboxylate isomerase protein family, which areinvolved in prokaryotic polyaromatic hydrocarbon (PAH) catabolism, haslow similarity to uncharacterized C. elegans ZK1320.1 53 7510146CD1g181333 1.3E−171 [Homo sapiens] steroid 11-beta-hydroxylase (Mornet, E.et al. (1989) J. Biol. Chem. 264 (35), 20961-20967) 53 7510146CD1709557|CYP11B1 2.8E−172 [Homo sapiens][Oxidoreductase; Smallmolecule-binding protein][Cytoplasmic; Mitochondrial] Steroid 11beta-hydroxylase, a cytochrome P450 that converts 11 deoxycortisol tocortisol; deficiency causes hypertensive congenital adrenal hyperplasia,and fusion of the gene with other genes is associated with diseases ofaldosterone synthesis. Pascoe, L. et al. Proc. Natl. Acad. Sci. U.S.A.89, 8327-8331 (1992). 53 7510146CD1 697979|Cyp11b2 9.2E−112 [Rattusnorvegicus][Oxidoreductase] Cytochrome P450 subfamily XIB polypeptide 2(aldosterone synthase), has 11-beta hydroxylase-aldosterone-2 synthaseactivity, expression is upregulated in fibrotic liver or by highpotassium or low sodium, may have a role in causing cardiac hypertrophy.Imai, M. et al. FEBS Lett. 263, 299-302 (1990).

TABLE 3 SEQ Incyte Potential ID Polypeptide Phosphorylation PotentialAnalytical Methods NO: ID Amino Acid Residues Sites Glycosylation SitesSignature Sequences, Domains and Motifs and Databases 1 7499940CD1 409S8 S74 S104 S105 3′5′-cyclic nucleotide phosphodiesterase: D155-R199HMMER_PFAM S121 S140 S145 S150 S152 S263 S320 S321 S351 S404 T25 T81T179 T194 T235 T252 T365 T388 PHOSPHODIESTERASE 4A CAMP CAMP-BLAST_PRODOM DEPENDENT 3′ 5′CYCLIC DPDE2 HYDROLASE ALTERNATIVE SPLICINGPD023907: D200-P408 CAMP-DEPENDENT 3′ 5′CYCLIC BLAST_PRODOMPHOSPHODIESTERASE HYDROLASE CAMP ALTERNATIVE SPLICING MULTIGENE FAMILYPD023901: G22-S89 PHOSPHODIESTERASE CAMP CAMP- BLAST_PRODOM DEPENDENT 3′5′CYCLIC HYDROLASE ALTERNATIVE SPLICING MULTIGENE FAMILY PD007678:F108-D155 3′5′-CYCLIC NUCLEOTIDE BLAST_DOMO PHOSPHODIESTERASESDM02037|P27815|1-245: M1-S245 DM07721|P27815|759-885: E282-T409BLAST_DOMO DM00370|P27815|343-722: D155-M246 BLAST_DOMODM00370|P14645|95-473: D155-E243 BLAST_DOMO 2 3329870CD1 418 S33 S86 S96S155 N220 N325 PROTEIN SIMILAR HUMAN DIHYDROXY BLAST_PRODOM S164 S198S222 VITAMIN D3INDUCED C04E12.11 BETA S241 S280 S358 ARRESTIN C04E12.12R06B9.3 PD004148: V23-A240 S399 S406 T132 T246 T271 T342 3 7500698CD1154 S20 T55 T100 NifU-like N terminal domain: L34-K147 HMMER_PFAM T106PROTEIN NIFU NITROGEN FIXATION OF BLAST_PRODOM PLASMID SECTION NIFU-LIKEGENE PRODUCT PD002743: Y35-Q144 NIFU; FIXATION; NITROGEN; YOR226C;BLAST_DOMO DM02171 |C64064|25-137: Y35-A132 BLAST_DOMO |S60953|24-137:R33-A132 BLAST_DOMO |P20628|1-118: V49-A132 BLAST_DOMO |P05343|1-112:Y35-A132 BLAST_DOMO 4 7500223CD1 363 S174 S217 S237 N263 N278 N332signal_cleavage: M1-G39 SPSCAN S284 S320 S343 T139 T166 T274 SignalPeptide: HMMER M22-A36, M22-G39, M22-A44, M22-L45 HMMER Arylesterase:HMMER_PFAM G23-L363 HMMER_PFAM Cytosolic domain: TMHMMER M1-R20 TMHMMERTransmembrane domain: TMHMMER A21-L43 TMHMMER Non-cytosolic domain:TMHMMER A44-L363 TMHMMER SERUM PARAOXONASE/ARYLES BLIMPS_PRODOM PD02637:R53-L107, E150-I178, T179-E226, BLIMPS_PRODOM G227-E257, V290-I315,Q316-L363 BLIMPS_PRODOM SERUM AROMATIC HYDROLASE BLIMPS_PRODOMGLYCOPROTEIN ESTERASE PARAOXONASE/ARYLESTERASE SIGNAL A- ESTERASEARYLDIAKYLPHOSPHATASE PD005046: E70-L363 BLAST_PRODOM SERUM AROMATICHYDROLASE BLIMPS_PRODOM GLYCOPROTEIN ESTERASE PARAOXONASE/ARYLESTERASESIGNAL A- ESTERASE ARYLDIAKYLPHOSPHATASE PD005529: M22-I69 BLAST_PRODOMSERUM PARAOXONASE/ARYLESTERASE BLAST_DOMO DM07178 BLAST_DOMOP54832|1-353: M22-L363 BLAST_DOMO P27169|1-353: R24-L363 BLAST_DOMO 57500295CD1 342 S153 S196 S216 N242 N257 N311 signal_cleavage: M1-G18SPSCAN S263 S299 S322 T118 T145 T253 Signal Peptide: HMMER M1-A15,M1-G18, M1-A23, M1-L24 HMMER Arylesterase: G2-L342 HMMER_PFAM SERUMPARAOXONASE/ARYLES BLIMPS_PRODOM PD02637: R32-L86, E129-I157, T158-E205,BLIMPS_PRODOM G206-E236, V269-I294, Q295-L342 BLIMPS_PRODOM SERUMAROMATIC HYDROLASE BLAST_PRODOM GLYCOPROTEIN ESTERASEPARAOXONASE/ARYLESTERASE SIGNAL A- ESTERASE ARYLDIAKYLPHOSPHATASEPD005046: E49-L342 BLAST_PRODOM SERUM AROMATIC HYDROLASE BLAST_PRODOMGLYCOPROTEIN ESTERASE PARAOXONASE/ARYLESTERASE SIGNAL A- ESTERASEARYLDIAKYLPHOSPHATASE PD005529: M1-I48 BLAST_PRODOM SERUMPARAOXONASE/ARYLESTERASE BLAST_DOMO DM07178 BLAST_DOMO P54832|1-353:M1-L342 BLAST_DOMO P27169|1-353: R3-L342 BLAST_DOMO 6 7502095CD1 416 S46S73 S94 S126 N253 Aminotransferase class I and II HMMER_PFAM S154 S325S390 T43 T51 T140 T235 T320 A90-V402 HMMER_PFAM Aminotransferasesclass-II pyridoxal-phosphate BLIMPS_BLOCKS attachment site BL00599:A65-S73, S93-A121, S147-I156, BLIMPS_BLOCKS D224-G236 BLIMPS_BLOCKSAminotransferases class-II pyridoxal-phosphate PROFILESCAN attachmentsite G236-Y285 PROFILESCAN 2-AMINO-3KETOBUTYRATE COA LIGASE ECBLAST_PRODOM 2.3.1.29 LIGASE TRANSFERASE ACYLTRANSFERASE PD168670:M1-I30 BLAST_PRODOM AMINOTRANSFERASES CLASS-II PYRIDOXAL- BLAST_DOMOPHOSPHATE ATTACHMENT SITE DM00464 BLAST_DOMO P07912|3-390: L31-G405BLAST_DOMO P53556|1-382: A65-G405 BLAST_DOMO P26505|1-394: F63-V404BLAST_DOMO P08262|1-393: I60-V404 BLAST_DOMO 7 7500507CD1 550 S365 S397S531 N47 N191 N225 signal_cleavage: M1-A15 SPSCAN T124 T195 T317 Y125Signal Peptide: HMMER M1-G17 HMMER Aminolevulinic acid synthase domain:HMMER_PFAM F106-R181 HMMER_PFAM Aminotransferase class I and II:HMMER_PFAM A184-L499 HMMER_PFAM Aminotransferases class-IIpyridoxal-phosphate BLIMPS_BLOCKS attachment site BL00599: D122-T130,G187-A215, S243-I252, BLIMPS_BLOCKS D320-G332, I345-T351 BLIMPS_BLOCKSAminotransferases class-II pyridoxal-phosphate PROFILESCAN attachmentsite: S330-F380 PROFILESCAN SYNTHASE ACID TRANSFERASE BLAST_PRODOMACYLTRANSFERASE 5-AMINOLEVULINIC DELTA-AMINOLEVULINATE DELTA-ALASYNTHETASE ERYTHROID-SPECIFIC MITOCHONDRIAL PRECURSOR PD013126: M1-T101BLAST_PRODOM SYNTHASE ACID TRANSFERASE 5- BLAST_PRODOM AMINOLEVULINICDELTA- AMINOLEVULINATE DELTA-ALA SYNTHETASE MITOCHONDRIAL PRECURSOR HEMEPD001038: L481-G542 BLAST_PRODOM SYNTHASE TRANSFERASE ACID SYNTHETASEBLAST_PRODOM BIOSYNTHESIS 5-AMINOLEVULINIC DELTA- AMINOLEVULINATEACYLTRANSFERASE DELTA-ALA HEME PD001058: Y138-S193 BLAST_PRODOM SYNTHASETRANSFERASE ACID DELTA- BLAST_PRODOM AMINOLEVULINATE 5-AMINOLEVULINICDELTA-ALA SYNTHETASE MITOCHONDRIAL PRECURSOR HEME PD003154: F106-A147BLAST_PRODOM AMINOTRANSFERASES CLASS-II PYRIDOXAL- BLAST_DOMO PHOSPHATEATTACHMENT SITE DM00464 BLAST_DOMO P22557|142-538: V105-A502 BLAST_DOMOP43090|138-533: F106-L500 BLAST_DOMO P07997|191-587: F106-L500BLAST_DOMO P43091|183-580: F106-W503 BLAST_DOMO Aminotransferasesclass-II pyridoxal-phosphate MOTIFS attachment site: T351-G360 MOTIFS 87500840CD1 142 S11 S83 T41 T42 Signal Peptide: M1-W24 HMMER FERREDOXIN[2FE-2S] BLAST_DOMO DM00144 BLAST_DOMO Q10361|517-620: V68-E131BLAST_DOMO S61012|59-162: V68-E131 BLAST_DOMO Adrenodoxin family,iron-sulfur binding region MOTIFS signature C105-H115 MOTIFS Cytochromec family heme-binding site signature MOTIFS C111-V116 MOTIFS 97493620CD1 524 S97 S131 S142 N66 N314 N477 Signal Peptide: M1-S18,M1-S21, M1-G23, M1-C22, HMMER S297 S416 T70 T81 M1-G20 T83 T205 T244T248 T503 Y235 UDP-glucoronosyl and UDP-glucosyl transferas: G23-K522HMMER_PFAM Cytosolic domain: TMHMMER Y511-E524 TMHMMER Transmembranedomain: TMHMMER V488-I510 TMHMMER Non-cytosolic domain: TMHMMER M1-D487TMHMMER UDP-glycosyltransferases proteins BLIMPS_BLOCKS BL00375:S33-L55, C126-P166, P189-N212, BLIMPS_BLOCKS I254-C281, F294-G343,N345-P389, BLIMPS_BLOCKS H444-Y483 BLIMPS_BLOCKSUDP-glycosyltransferases signature PROFILESCAN N373-T414 PROFILESCANTRANSFERASE GLYCOSYLTRANSFERASE BLAST_PRODOM PROTEIN UDP-GLUCURONOSYLTRANSFERASE PRECURSOR SIGNAL TRANSMEMBRANE UDP-GTGLYCOPROTEIN MICROSOMAL PD000190: G23-T324, I386-E524, S297-P431BLAST_PRODOM UDP-GLUCORONOSYL AND UDP-GLUCOSYL BLAST_DOMO TRANSFERASESDM00367 BLAST_DOMO P36537|186-460: F186-F457 BLAST_DOMO P17717|188-462:F186-F457 BLAST_DOMO P16662|187-461: F186-F457 BLAST_DOMOP36538|187-461: I187-F457 BLAST_DOMO 10 7494697CD1 300 S20 S95 S198 S207N246 Zinc-binding dehydrogenases: HMMER_PFAM T8 T18 T202 Y296 D21-D300HMMER_PFAM NADP-DEPENDENT OXIDOREDUCTASE NADP BLAST_PRODOM PROTEINLEUKOTRIENE B4 12HYDROXYDEHYDROGENASE PROBABLE 15- OXOPROSTAGLANDIN13-REDUCTASE PD005709: R3-R51 BLAST_PRODOM ZINC-CONTAINING ALCOHOLBLAST_DOMO DEHYDROGENASES DM00064 BLAST_DOMO S47093|9-327: L9-D300BLAST_DOMO S57611|3-340: L9-E293 BLAST_DOMO S58197|17-359: F22-N246BLAST_DOMO S57614|290-616: V68-Y245 BLAST_DOMO 11 8146738CD1 483 S89S112 S194 N409 N453 signal_cleavage: M1-A21 SPSCAN S394 S424 S431 T67T383 T450 Y337 Signal Peptide: HMMER M1-A16, M1-I18, M1-A21, M1-Q23HMMER Glycosyl hydrolases family: HMMER_PFAM Y22-D366 HMMER_PFAMChitinases family 18 proteins BLIMPS_BLOCKS BL01095: G98-T108,F133-G144, F355-D366 BLIMPS_BLOCKS HYDROLASE GLYCOSIDASE PROTEINBLAST_PRODOM CHITINASE PRECURSOR SIGNAL GLYCOPROTEIN CHITIN DEGRADATIONENDOCHITINASE PD000471: T29-S322, E168-D366 BLAST_PRODOM CHITINASESFAMILY 18 proteins BLAST_DOMO DM00467 BLAST_DOMO S27879|27-365: Y27-D366BLAST_DOMO P36222|27-356: Y27-D366 BLAST_DOMO S51327|27-356: Y27-D366BLAST_DOMO I48271|27-357: Y27-D366 BLAST_DOMO Chitinases family 18active site: MOTIFS F133-E141 MOTIFS 12 7500114CD1 254 S17 S69 S78 S130Signal Peptide: M4-S25, M4-G27, M1-G27 HMMER S183 S244 T118 T251HMGL-like: HMMER_PFAM R41-V247 HMMER_PFAM Hydroxymethylglutaryl-coenzymeA lyase proteins BLIMPS_BLOCKS BL01062: T107-I142, M143-D186, S187-G232BLIMPS_BLOCKS HYDROXYMETHYLGLUTARYLCOA LYASE BLAST_PRODOM PRECURSORHMGCOA HL 3HYDROXY3METHYLGLUTARATECOA MITOCHONDRION TRANSIT PEPTIDEDISEASE PD023169: M1-P40 BLAST_PRODOM LYASE SYNTHASE PYRUVATE 2-BLAST_PRODOM ISOPROPYLMALATE CARBOXYLASE BIOTIN PROTEIN HOMOCITRATEBIOSYNTHESIS ALPHA-ISOPROPYLMALATE PD000608: V117-I235, R41-E72BLAST_PRODOM HYDROXYMETHYLGLUTARYL-COENZYME A BLAST_DOMO LYASE DM08710BLAST_DOMO P35915|3-297: A115-L254, P30-L131 BLAST_DOMO P13703|1-300:A115-A250, V33-L131 BLAST_DOMO Hydroxymethylglutaryl-coenzyme A lyaseactive site: MOTIFS S188-Y197 MOTIFS Prenylation: MOTIFS C252-L254MOTIFS 13 7500197CD1 374 S29 S34 S46 S64 signal_cleavage: M1-C51 SPSCANT95 T177 T255 Y117 Y259 Signal Peptide: HMMER M1-A21 HMMER Polyprenylsynthetase: HMMER_PFAM R110-Q337 HMMER_PFAM Polyprenyl synthetasesproteins BLIMPS_BLOCKS BL00723: G121-V131, D169-C183, T255-M280,BLIMPS_BLOCKS M301-K323 BLIMPS_BLOCKS FARNESYL PYROPHOSPHATE SYNTHETASEBLAST_PRODOM FPP FPS DIPHOSPHATE INCLUDES: DIMETHYLALLYLTRANSFERASEGERANYLTRANSTRANSFERASE TRANSFERASE PD122945: M67-R110 BLAST_PRODOMPOLYPRENYL SYNTHETASES BLAST_DOMO DM00371 BLAST_DOMO P14324|7-267:S73-Y311 BLAST_DOMO B34713|7-267: D74-Y311 BLAST_DOMO P08524|2-264:K80-Y311 BLAST_DOMO P49349|2-261: A77-Y311 BLAST_DOMO Polyprenylsynthetases signature 1: MOTIFS L166-G174 MOTIFS 14 7500145CD1 327 S103S115 S187 N60 signal_cleavage: M1-A21 SPSCAN S277 T82 Y189 SignalPeptides: M1-A21, M1-L24, M1-C26 HMMER Glycosyl hydrolases family 18:V199-D301, Y22-L198 HMMER_PFAM Chitinases family 18 proteinsBLIMPS_BLOCKS BL01095: G97-S107, F132-G143, L290-D301 HYDROLASEGLYCOSIDASE PROTEIN BLAST_PRODOM CHITINASE PRECURSOR SIGNAL GLYCOPROTEINCHITIN DEGRADATION ENDOCHITINASE PD000471: Y22-F205, L198-D301, Y22-I61CARTILAGE GLYCOPROTEIN 39 39 KD BLAST_PRODOM SYNOVIAL PROTEIN YKL40CHITINASE 3 LIKE 1 GLYCOPROTEIN SIGNAL PD164290: S30-I66 CHITINASESFAMILY 18 BLAST_DOMO DM00467|P36222|27-356: Y27-F205, L198-D301DM00467|S51327|27-356: Y27-L198, L198-D301 DM00467|I48271|27-357:Y27-L198, L198-D301 DM00467|S61550|27-357: Y27-L198, L198-D301 157500874CD1 207 S103 S115 S122 N60 signal_cleavage: M1-A21 SPSCAN S157T82 Signal Peptides: M1-A21, M1-L24, M1-C26 HMMER Glycosyl hydrolasesfamily 18: G129-D181, Y22-R128 HMMER_PFAM CARTILAGE GLYCOPROTEIN 39 39KD BLAST_PRODOM SYNOVIAL PROTEIN YKL40 CHITINASE3 LIKE 1 GLYCOPROTEINSIGNAL PD164290: S30-I66 HYDROLASE GLYCOSIDASE PROTEIN BLAST_PRODOMCHITINASE PRECURSOR SIGNAL GLYCOPROTEIN CHITIN DEGRADATION ENDOCHITINASEPD000471: Y22-D167, P141-D181, Y22-I61 CHITINASES FAMILY 18 BLAST_DOMODM00467|S61550|27-357: Y27-R128, I123-D181 DM00467|I48271|27-357:Y27-R128, I123-D181 DM00467|P36222|27-356: Y27-Q169, I123-D181DM00467|S51327|27-356: Y27-Q148, I123-D181 16 7500495CD1 169 S34 S82S137 signal_cleavage: M1-A28 SPSCAN 17 7500194CD1 360 S21 S199 S205 N222N349 Signal Peptide: M6-G29 HMMER T329 T340 Acyl-CoA dehydrogenase,N-terminal domain: HMMER_PFAM W111-A191 Acyl-CoA dehydrogenase, middledomain: HMMER_PFAM C193-L301 Acyl-CoA dehydrogenases proteinsBLIMPS_BLOCKS BL00072: L117-E127, Y219-G231, G268-F308 Acyl-CoAdehydrogenases signatures: L194-T250 PROFILESCAN PROTEIN DEHYDROGENASEACYL-CoA BLAST_PRODOM OXIDOREDUCTASE FLAVOPROTEIN FAD OXIDASE FATTY ACIDMETABOLISM PD000396: V71-T285, V71-A357 ACYL-CoA DEHYDROGENASE VERY LONGBLAST_PRODOM CHAIN SPECIFIC PRECURSOR VLCAD OXIDOREDUCTASE FLAVOPROTEINFAD FATTY PD015520: M1-Q46, A44-V71 ACYL-COA DEHYDROGENASES BLAST_DOMODM00853|P48818|85-478: D63-V338 DM00853|P45857|1-377: L72-A357DM00853|P45867|3-379: L72-E343 DM00853|Q06319|3-383: L114-V338 Acyl-CoAdehydrogenases signature 1: C193-S205 MOTIFS 18 7500871CD1 305 S25 S37S109 S157 Glycosyl hydrolases family 18: M1-D279 HMMER_PFAM S255 T4 Y111Chitinases family 18 proteins BLIMPS_BLOCKS BL01095: G19-S29, F54-G65,L268-D279 HYDROLASE GLYCOSIDASE PROTEIN BLAST_PRODOM CHITINASE PRECURSORSIGNAL GLYCOPROTEIN CHITIN DEGRADATION ENDOCHITINASE PD000471: K6-T229,H140-D279, D55-K80 CHITINASES FAMILY 18 BLAST_DOMODM00467|P36222|27-356: M1-D279 DM00467|S51327|27-356: L2-D279DM00467|I48271|27-357: L2-D279 DM00467|S61550|27-357: L2-D279 Sugartransport proteins signature 2: F130-R155 MOTIFS 19 7500873CD1 227 S31S79 S177 Y33 signal_cleavage: M1-T28 SPSCAN Glycosyl hydrolases family18: M1-D201 HMME_PFAM HYDROLASE GLYCOSIDASE PROTEIN BLAST_PRODOMCHITINASE PRECURSOR SIGNAL GLYCOPROTEIN CHITIN DEGRADATION ENDOCHITINASEPD000471: K13-T151, H62-D201 CHITINASES FAMILY 18 BLAST_DOMODM00467|P36222|27-356: M1-D201 DM00467|S51327|27-356: M1-D201DM00467|I48271|27-357: M1-D201 DM00467|S61550|27-357: M1-D201 Sugartransport proteins signature 2: F52-R77 MOTIFS 20 7503491CD1 346Potential Potential Uroporphyrinogen decarboxylase (URO-D): L14-H339HMMER_PFAM Phosphorylation Glycosylation Sites: Sites: S86 S292 N16 T58Uroporphyrinogen decarboxylase proteins BL00906: BLIMPS_BLOCKSL280-Y290, R311-L320, F19-Y42, R127-P164, Q165-F208 UROPORPHYRINOGENDECARBOXYLASE BLAST_PRODOM LYASE PORPHYRIN BIOSYNTHESIS UPDMETHYLTRANSFERASE TRANSFERASE HEME A PD003225: Q71-H337, K15-L73UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMO DM01567 P06132|11-366:Q71-N346, F11-L73 P32347|4-361: Q71-K338, F11-Q71 P29680|1-353:L68-S340, E13-L73 P32395|3-352: L70-R341, E13-T69 Atp_Gtp_A: A270-T277MOTIFS Urod_1: P32-R41 MOTIFS Urod_2: G132-G147 MOTIFS 21 7503427CD1 193Potential Potential NAD(P)H dehydrogenase (quinone): D41-Q175 HMMER_PFAMPhosphorylation Glycosylation Sites: Sites: S21 S61 S159 N19 S171 T38T52 OXIDOREDUCTASE NADPH PROTEIN BLAST_PRODOM PUTATIVE DEHYDROGENASEQUINONE REDUCTASE AZOREDUCTASE PHYLLOQUINONE MENADIONE PD004598:G102-E180 NADPH DEHYDROGENASE QUINONE BLAST_PRODOM REDUCTASEAZOREDUCTASE PHYLLOQUINONE MENADIONE OXIDOREDUCTASE NAD NADP PD016667:M1-Y68 NADPH DEHYDROGENASE QUINONE 2 EC BLAST_PRODOM 1.6.99.2 REDUCTASEDTDIAPHORASE AZOREDUCTASE PHYLLOQUINONE MENADIONE OXIDOREDUCTASE NADNADP FLAVOPROTEIN FAD MULTIGENE FAMILY PD099728: M166-Q193 NAD;OXIDOREDUCTASE; DEHYDROGENASE; BLAST_DOMO SPOIIIC;DM02281|P16083|39-219: D96-P182, V39-V109 NAD; OXIDOREDUCTASE;DEHYDROGENASE; BLAST_DOMO SPOIIIC; DM02281|P15559|39-219: F100-P182,S40-V109 22 7503547CD1 178 Potential Signal_cleavage: M1-A64 SPSCANPhosphorylation Sites: S162 T172 Short-chain dehydrogenases/reductasesfamily PROFILESCAN signature: G98-V152 DIHYDROPTERIDINE REDUCTASE HDHPRBLAST_PRODOM QUINOID TETRAHYDROBIOPTERIN BIOSYNTHESIS OXIDOREDUCTASENADP 3DSTRUCTURE PHENYLKETONURIA PD038408: V36-V178, G8-L53 A55R;REDUCTASE; TERMINAL; BLAST_DOMO DIHYDROPTERIDINE; DM00099|P09417|78-113:E47-T83 Adh_Short: A106-A134 MOTIFS 23 1932641CD1 556 PotentialPotential Signal_cleavage: M1-P63 SPSCAN Phosphorylation GlycosylationSites: Sites: S35 S49 S102 N213 N236 N390 S143 S175 S313 T243 T333 T374T402 Y352 O-PHOSPHATIDYL-TRANSFERASE CDP- BLAST_PRODOMDIACYLGLYCEROLSERINE PHOSPHATIDYLSERINE SYNTHASE PHOSPHOLIPIDBIOSYNTHESIS MEMBRANE PUTATIVE MITOCHONDRION PD014389: N85-L522 PEL1;SYNTHASE; PHOSPHATIDYLSERINE; BLAST_DOMO DM05669|P25578|1-145: R84-N213PHOSPHATIDYLTRANSFERASE; BLAST_DOMO DIACYLGLYCEROL; CDP;CDPDIACYLGLYCEROL; DM07147|P44704|1-454: N85-E298, M308-F555 246892447CD1 1558 Potential AMP-binding enzyme: T1005-V1477, T353-I499,HMMER_PFAM Glycosylation Sites: V706-R805 N205 N494 N612 N1383SIMILARITY TO AN AMP-BINDING MOTIF BLAST_PRODOM PD147817: L645-C1006;PD170422: F1478-M1558, I842-V914 CHROMOSOME PROTEIN I TRANSMEMBRANEBLAST_PRODOM YOR3170C FROM XV C22F3.04 C56F8.02 PD016696: S1260-L1540SPAC22F3.04; DM05110|Q10250|778-1480: H872-Y1556, BLAST_DOMO T341-E847SPAC22F3.04; DM05110|S62419|703-1389: H1031-R1537 BLAST_DOMOSPAC22F3.04; DM05110|Q09773|693-1389: H1031-R1537 BLAST_DOMO MASC;DM08837|Q10976|56-610: Q979-R1537, BLAST_DOMO P382-S898, A846-G894Potential Phosphorylation Sites: S31 S81 S82 S84 MOTIFS S116 S120 S137S139 S253 S257 S333 S361 S615 S631 S655 S717 S802 S852 S947 S955 S1165S1210 S1236 S1247 S1251 S1313 S1348 S1406 S1471 S1493 S1531 T12 T125T131 T201 T266 T340 T374 T420 T503 T533 T668 T702 T853 T984 T1058 T1073Crystallin_Betagamma: I1043-T1058 MOTIFS 25 7503416CD1 608 PotentialPhosphoenolpyruvate carboxykinase: D46-P456, HMMER_PFAM PhosphorylationK457-M608 Sites: S23 S51 S115 S136 S187 S535 T29 T66 T75Phosphoenolpyruvate carboxykinase (GTP) proteins BLIMPS_BLOCKS BL00505:G339-A365, A367-E389, W404-I446, P441-L484, P495-G532, K88-P121,G132-G175, V176-G195, D204-P217, W228-L258, L266-L318Phosphoenolpyruvate carboxykinase (GTP) signature: PROFILESCAN H282-I330PHOSPHOENOLPYRUVATE CARBOXYKINASE BLAST_PRODOM GTP CARBOXYLASE LYASEDECARBOXYLASE GTP-BINDING GLUCONEOGENESIS PEPCK CYto SOLIC PD004738:D46-K457, K457-M608 PHOSPHOENOLPYRUVATE CARBOXYKINASE, BLAST_PRODOMMITOCHONDRIAL PRECURSOR GTP EC 4.1.1.32 CARBOXYLASE PEPCKMGLUCONEOGENESIS LYASE DECARBOXYLASE GTP-BINDING MITOCHONDRION TRANSITPEPTIDE MANGANES PD144568: M1-R45 PHOSPHOENOLPYRUVATE CARBOXYKINASEBLAST_DOMO (GTP) DM01781 P05153|15-621: V32-F466, K457-M608P20007|40-646: G35-D464, K457-M608 P29190|9-617: G35-G458, K457-K607Q05893|30-640: V32-G458, G458-V605 Pepck_Gtp: F302-N310 MOTIFS 267503874CD1 450 Potential Potential Cyto Solic domain: M1-S37 TMHMMERPhosphorylation Glycosylation Sites: Transmembrane domain: L38-I60Sites: S10 S14 S33 N220 N284 Non-cyto Solic domain: K61-S450 S37 S238S301 S317 S395 T9 T93 T134 T286 T420 Signal_cleavage: M29-A77 SPSCANGDA1_CD39 GDA1/CD39 (nucleoside phosphatase) HMMER_PFAM family GDA1/CD39family of nucleoside phosphatases BLIMPS_BLOCKS proteins BL01238:G248-F261, I104-F118, P176-R186, M219-K240 CD39L2 PD175837: V310-S450;PD172427: M1-G97 BLAST_PRODOM HYDROLASE TRANSMEMBRANE PROTEINBLAST_PRODOM NUCLEOSIDE CD39 NUCLEOSIDETRIPHOSPHATASE TRIPHOSPHATENTPASE PRECURSOR ATPDIPHOSPHOHYDROLASE PD003822: V100-S293, E191-V310,F394-G433 ACTIVATION; NUCLEOSIDE; ANTIGEN; BLAST_DOMO LYMPHOID; DM02628P32621|84-517: T93-R303, N332-A434 P52914|35-454: Y102-L307, F345-L442P40009|1-462: T134-R303, Y102-T134, K422-Y438 I56242|40-471: V100-G29827 7503454CD1 209 Potential Potential Glutathione S-transferase,N-terminal domain: E21-D95 HMMER_PFAM Phosphorylation GlycosylationSites: Sites: S28 S35 S138 N128 Y64 Glutathione S-transferase PF000043:I72-S101 BLIMPS_PFAM 28 7503528CD1 214 Potential2-hydroxychromene-2-carboxylate isomer: T7-E200 HMMER_PFAMPhosphorylation Sites: S188 T149 Y12 ISOMERASE PROTEIN S-TRANSFERASEBLAST_PRODOM CHROMOSOME DIOXYGENASE 2HYDROXYCHROMENE2-CARBOXYLATEPLASMID THE GLUTATHIONE MITOCHONDRIAL PD008447: R6-G199 29 7503705CD1332 S59 S184 S189 T34 N152 N221 signal_cleavage: M1-P23 SPSCAN Y103 Y214Signal Peptides: M1-C18, M1-G21, M1-P23, M1-C24, HMMER M1-C28, M1-P20,M1-S26 von Willebrand factor type C domain: C264-C319 HMMER_PFAMPEROXIDASE OXIDOREDUCTASE PRECURSOR BLAST_PRODOM SIGNAL HEMEGLYCOPROTEIN PROTEIN SIMILAR MYELOPEROXIDASE EOSINOPHIL PD001354:L56-F141 MYELOPEROXIDASE BLAST_DOMO DM01034|S46224|911-1352: L56-C167DM01034|P11678|282-714: L56-Q165 DM01034|P05164|310-743: Y57-D166DM01034|B28894|395-828: Y57-D166 VWFC domain signature: C283-C319 MOTIFS30 7503707CD1 1316 S90 S167 S171 N271 N387 N401 signal_cleavage: M1-P23SPSCAN S233 S310 S500 N529 N626 N705 Signal Peptides: M1-C18, M1-G21,M1-P23, M1-C24, HMMER S554 S613 S627 N717 N1068 N1161 M1-C28, M1-P20,M1-S26 S634 S696 S719 N1283 Animal haem peroxidase: K726-Q1265HMMER_PFAM S871 S903 S929 Leucine Rich Repeat: R147-D170, Q51-K74,S123-L146, HMMER_PFAM S1164 S1190 T34 N75-E98, N99-I122 T53 T117 T141Leucine rich repeat C-terminal domain: N180-Q232 HMMER_PFAM T225 T254T347 T389 T424 T472 Immunoglobulin domain: G344-A400, G248-A307,HMMER_PFAM T504 T520 T566 G525-A582, C440-A490 T628 T639 T710 Animalhaem peroxidase signature PR00457: R751-R762, BLIMPS_PRINTS T823 T1070T1123 M802-T817, F954-T972, T972-W992, V997-G1023, Y303 Y1234T1050-I1060, D1177-W1197, L1248-D1262 PEROXIDASE OXIDOREDUCTASEPRECURSOR BLAST_PRODOM SIGNAL HEME GLYCOPROTEIN PROTEIN SIMILARMYELO-PEROXIDASE EOSINOPHIL PD001354: K1166-F1272 PROTEIN ZK994.3K09C8.5 PEROXIDASIN BLAST_PRODOM PRECURSOR SIGNAL PD144227: N584-K726PEROXIDASE OXIDOREDUCTASE PRECURSOR BLAST_PRODOM SIGNAL MYELOPEROXIDASEHEME GLYCOPROTEIN ASCORBATE CATALASE LASCORBATE PD000217: Y727-A784,F1086-T1163, R825-K931 HEMICENTIN PRECURSOR SIGNAL BLAST_PRODOMGLYCOPROTEIN EGF-LIKE DOMAIN HIM4 PROTEIN ALTERNATIVE SPLICING PD066634:P234-C398, N401-C580 MYELOPEROXIDASE BLAST_DOMO DM01034|S46224|911-1352:C859-C1298 DM01034|P09933|284-735: A857-D1297 DM01034|P35419|276-725:C859-D1297 DM01034|P11678|282-714: F862-Q1296 31 90001962CD1 449 S88S198 S218 N156 N194 Signal Peptide: M1-Q22 HMMER S271 S298 S379Cytochrome P450: W264-L412, P29-M73 HMMER_PFAM S389 S418 T77 Cytosolicdomain: Q22-G247 TMHMMER T104 T162 T238 Transmembrane domains: I4-L21,L248-L270 T315 T325 Y173 Non-cytosolic domains: M1-L3, S271-I449 Y337E-class P450 group I signature PR00463: R57-A76, BLIMPS_PRINTSA262-G288, S348-K372, F384-C394, C394-C417 E-class P450 group IIsignature PR00464: L50-G70, BLIMPS_PRINTS S271-G288, K304-I324,G342-K357, Y358-A373, L381-C394, C394-C417 E-class P450 group IVsignature PR00465: P29-G46, BLIMPS_PRINTS E51-T74, P244-L270, L305-P321,Y337-W351, H353-K371, H378-C394, C394-L412 CYTOCHROME P450 BLAST_DOMODM00022|S50211|59-488: W252-E438 DM00022|S45039|89-486: A253-L419DM00022|P51538|59-488: L112-E438 DM00022|P24462|59-488: Y147-Y415 3270819231CD1 711 S6 S12 S24 S35 N200 N301 DDHD domain: L495-Q700HMMER_PFAM S73 S367 S373 SAM domain (Sterile alpha motif): D383-K445HMMER_PFAM S442 S447 S489 WWE domain: S35-R112 HMMER_PFAM S593 S624 S626PROTEIN CHROMOSOME PHOSPHATIDIC ACID BLAST_PRODOM S670 T114 T145PREFERRING PHOSPHOLIPASE A1 SIMILARITY T184 T193 T279 OVER A SHORTPD014530: F267-Q364, L653-E697, T303 T318 T389 C530-L586, I213-S243 337504066CD1 236 S13 S52 S102 S189 signal_cleavage: M1-F18 SPSCAN T57 T158NAD(P)H dehydrogenase (quinone): D41-E175 HMMER_PFAM Ribosomal proteinS5 signature: I50-S114 PROFILESCAN NADPH DEHYDROGENASE QUINONEBLAST_PRODOM REDUCTASE AZOREDUCTASE PHYLLOQUINONE MENADIONEOXIDOREDUCTASE NAD NADP PD022346: S154-K236 PD016667: M1-Y68OXIDOREDUCTASE NADPH PROTEIN BLAST_PRODOM PUTATIVE DEHYDROGENASE QUINONEREDUCTASE AZOREDUCTASE PHYLLOQUINONE MENADIONE PD004598: K103-Y153,A75-Q101 NAD; OXIDOREDUCTASE; DEHYDROGENASE; BLAST_DOMO SPOIIIC;DM02281|P15559|39-219: F66-P182, E39-Q101 DM02281|P16083|39-219:D96-P182, S40-Q101 34 90001862CD1 598 S32 S36 S63 S138 N43 N136 Rieske[2Fe—2S] domain: V68-S168 HMMER_PFAM S219 S300 S305 S359 S414 S521 S576T45 T212 T244 T277 T316 T319 T352 T550 T594 Y164 Pyridinenucleotide-disulphide oxidoreductase: N196-N478 HMMER_PFAM FAD-dependentpyridine nucleotide reductase BLIMPS_PRINTS signature PR00368:L293-K302, N334-S359, D421-F435, V462-V469, N196-F218 Pyridinenucleotide disulphide reductase class-II BLIMPS_PRINTS signaturePR00469: N196-F218, A330-K354, R388-E404, V422-L443, T457-W475IRON-SULFUR ELECTRON TRANSPORT BLIMPS_PRODOM PD02042: V93-G119,V126-G140 TAMEGOLOH PD067039: M1-A71 BLAST_PRODOM PROTEIN TAMEGOLOH EG:22E5.5 PUTATIVE BLAST_PRODOM FLAVOPROTEIN C26F1.14C SIMILAROXIDOREDUCTASE PD020901: Y512-E586 OXIDOREDUCTASE FLAVOPROTEIN FADBLAST_PRODOM REDUCTASE REDOXACTIVE CENTER DEHYDROGENASE PROTEIN NADP NADPD000139: L288-D421, V418-E506, D77-L95 PYRIDINE NUCLEOTIDE-DISULPHIDEBLAST_DOMO OXIDOREDUCTASES CLASS-I DM00071 |P17052|1-243: V197-P431|P43494|1-242: N196-P431 |Q07946|1-243: S194-A432 |P37337|1-243:V197-A432 35 7503046CD1 435 S93 S189 S218 signal_cleavage: M1-S43 SPSCANS242 S335 S381 S401 T142 T396 Thioredoxin family proteins BL00194:G197-R209 BLIMPS_BLOCKS Thioredoxin family signature PR00421: V196-W204,BLIMPS_PRINTS W204-R213, G271-D282 PROTEIN ANTIOXIDANT PEROXIDASEBLIMPS_PRODOM PD00210: V196-L211 NUCLEOREDOXIN RED1 GENE PD084980:H308-I435 BLAST_PRODOM NUCLEOREDOXIN RED1 GENE PD077508: M1-Q101BLAST_PRODOM PROTEIN REDOXACTIVE CENTER T13D8.29 BLAST_PRODOMTRYPAREDOXIN NUCLEOREDOXIN RED1 GENE PREDICTED II PD150301: Y246-W307,D102-W165 PROTEIN T13D8.29 REDOXACTIVE CENTER BLAST_PRODOM THIOREDOXINC35B1.5 R05H5.3 COSMID F29B9 F17B5.1 PD004855: S190-Y246 36 7503211CD1437 S249 S350 T71 signal_cleavage: M1-A23 SPSCAN T326 T372 T411 T432Cytochrome P450: P42-G400, V401-A435 HMMER_PFAM Mitochondrial P450signature PR00408: F193-L211, BLIMPS_PRINTS R332-L345, T360-V378,W116-L131 E-class P450 group II signature PR00464: H194-V212,BLIMPS_PRINTS A303-A331, R332-A349, E361-F381 CYTOCHROME P450 ELECTRONTRANSPORT BLAST_PRODOM OXIDOREDUCTASE PRECURSOR MONOOXYGENASE MEMBRANEHEME STEROID PD002412: M1-W49 CYTOCHROME P450 DM00022 BLAST_DOMO|P15538|84-494: G84-L402 |P19099|84-494: G84-L402 |P15150|83-494:L83-L402 |P30099|94-501: G84-L402 37 7503264CD1 271 S5 S204 T82 T214N226 Inorganic pyrophosphatase: H27-A211 HMMER_PFAM T228 T260 Inorganicpyrophosphatase proteins BL00387: F26-M40, BLIMPS_BLOCKS D54-K91,G115-D145 Inorganic pyrophosphatase signature: A78-G124 PROFILESCANINORGANIC PYROPHOSPHATASE EC 3.6.1.1 BLAST_PRODOM PYROPHOSPHATE PHOSPHOHYDROLASE PPASE MAGNESIUM PD095166: L212-N271 INORGANIC PYROPHOSPHATASEBLAST_PRODOM PYROPHOSPHATE PPASE HYDROLASE MAGNESIUM PHOSPHO SOLUBLEPROTEIN PHOSPHOHYDROLASE PD002014: H27-A211 INORGANIC PYROPHOSPHATASEDM0100 BLAST_DOMO |P37980|33-227: V19-K210 |P13998|29-227: V25-K210|P28239|62-260: H27-D207 |P19117|31-228: K23-K210 Inorganicpyrophosphatase signature: D98-V104 MOTIFS 38 90120235CD1 341 S95 S118S239 N99 N236 signal_cleavage: M1-D58 SPSCAN S252 T26 T101 T198 T201T250 T268 T300 39 90014961CD1 314 S44 S86 S164 S247 N100 N311signal_cleavage: M1-G14 SPSCAN T78 T95 T269 Y112 Glycerophosphoryldiester phosphodiesterase: H45-R306 HMMER_PFAM Cytosolic domain:K25-L199 TMHMMER Transmembrane domains: A5-L24, F200-I22 Non-cytosolicdomains: M1-T4, R223-A314 PROTEIN HYDROLASE PHOSPHODIESTERASEBLAST_PRODOM GLYCEROPHOSPHORYL DIESTER GLYCEROPHOSPHODIESTER GLYCEROLMETABOLISM PRECURSOR CHROMOSOME PD002136: I43-K153 PHOSPHODIESTERASE;BLAST_DOMO GLYCEROPHOSPHORYL; DIESTER; MEMBRANE; DM01508|P54527|1-159:L39-C189 40 7503199CD1 271 S8 S74 S104 S105 PHOSPHODIESTERASE 4A cAMPcAMP- BLAST_PRODOM S125 S182 S183 cAMP-DEPENDENT 3′ 5′ CYCLICBLAST_PRODOM S213 S266 T25 T81 PHOSPHODIESTERASE HYDROLASE cAMP T114T227 T250 ALTERNATIVE SPLICING MULTIGENE FAMILY PD023901: G22-S893′5′-CYCLIC NUCLEOTIDE BLAST_DOMO PHOSPHODIESTERASESDM07721|P27815|759-885: E144-T271 DM02037|P27815|1-245: M1-S213DM07721|P14645|475-609: Q169-P270 41 7511530CD1 102 N16 Signal_cleavage:M1-C54 SPSCAN UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMODM01567|P06132|11-366: F11-P44 Uroporphyrinogen decarboxylase signature1: P32-R41 MOTIFS 42 7511535CD1 328 S274 T58 N16 Uroporphyrinogendecarboxylase (URO-D): L14-H321 HMMER_PFAM Uroporphyrinogendecarboxylase (URO-D) BLIMPS_BLOCKS IPB000257: L20-A39, C59-Q104,P111-P146, Q147-Y197, R293-L302, V240-I278 UROPORPHYRINOGENDECARBOXYLASE BLAST_PRODOM LYASE PORPHYRIN BIOSYNTHESIS UPDMETHYLTRANSFERASE TRANSFERASE HEME A PD003225: K15-R74 P72-H319UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMO DM01567|P06132|11-366:F11-R74, Q71-N328 UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMODM01567|P29680|1-353: E13-R74, P72-S322 UROPORPHYRINOGEN DECARBOXYLASEBLAST_DOMO DM01567|P32347|4-361: Q71-K320, F11-P111 UROPORPHYRINOGENDECARBOXYLASE BLAST_DOMO DM01567|P32395|3-352: E13-E75, Q71-R323ATP/GTP-binding site motif A (P-loop): A252-T259 MOTIFS Uroporphyrinogendecarboxylase signature 1: P32-R41 MOTIFS Uroporphyrinogen decarboxylasesignature 2: G114-G129 MOTIFS 43 7511536CD1 313 S107 S259 T58 N16Uroporphyrinogen decarboxylase (URO-D): L14-H306 HMMER_PFAMUroporphyrinogen decarboxylase (URO-D) BLIMPS_BLOCKS IPB000257: L20-A39,C59-K104, V225-I263, R278-L287 UROPORPHYRINOGEN DECARBOXYLASEBLAST_PRODOM LYASE PORPHYRIN BIOSYNTHESIS UPD METHYLTRANSFERASETRANSFERASE HEME A PD003225: K15-P158, A155-H304 UROPORPHYRINOGENDECARBOXYLASE BLAST_DOMO DM01567|P06132|11-366: F11-P158 V149-N313UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMO DM01567|P32347|4-361: F11-I183A155-K305 UROPORPHYRINOGEN DECARBOXYLASE BLAST_DOMODM01567|P32395|3-352: E13-P158 A155-R308 UROPORPHYRINOGEN DECARBOXYLASEBLAST_DOMO DM01567|P16891|2-353: L20-P158 G156-L310 ATP/GTP-binding sitemotif A (P-loop): A237-T244 MOTIFS Uroporphyrinogen decarboxylasesignature 1: P32-R41 MOTIFS 44 7511583CD1 162 S59 T156 DIHYDROPTERIDINEREDUCTASE HDHPR BLAST_PRODOM QUINOID TETRAHYDROBIOPTERIN BIOSYNTHESISOXIDOREDUCTASE NADP 3DSTRUCTURE PHENYLKETONURIA PD038408: G8-P145 A55R;REDUCTASE; TERMINAL; BLAST_DOMO DIHYDROPTERIDINE; DM00099|P09417|78-113:E78-T114 45 7511395CD1 444 S97 S131 S142 N66 N230 N397 Signal Peptide:M1-S18 HMMER S213 S336 S352 T70 T81 T83 T160 T164 Signal Peptide: M1-S21HMMER Signal Peptide: M1-G23 HMMER Signal Peptide: M1-C22 HMMER SignalPeptide: M1-G20 HMMER UDP-glucoronosyl and UDP-glucosyl transferas:G23-K442 HMMER_PFAM Cytosolic domain: K433-D444; Transmembrane TMHMMERdomain: G410-W432; Non-cytosolic domain: M1-I409 UDP-glucoronosyl andUDP-glucosyl transferase BLIMPS_BLOCKS IPB002213: W271-D313UDP-glycosyltransferases signature: N293-T334 PROFILESCAN TRANSFERASEGLYCOSYLTRANSFERASE BLAST_PRODOM PROTEIN UDPGLUCURONOSYLTRANSFERASEPRECURSOR Signal TRANSMEMBRANE UDPGT GLYCOPROTEIN MICROSOMAL PD000190:G23-G156, V211-S352, S336-R443, I61-L262 UDP-GLUCORONOSYL ANDUDP-GLUCOSYL BLAST_DOMO TRANSFERASES DM00367|P36537|186-460: G156-F377UDP-GLUCORONOSYL AND UDP-GLUCOSYL BLAST_DOMO TRANSFERASESDM00367|P16662|187-461: G156-F377 UDP-GLUCORONOSYL AND UDP-GLUCOSYLBLAST_DOMO TRANSFERASES DM00367|P36538|187-461: G156-F377UDP-GLUCORONOSYL AND UDP-GLUCOSYL BLAST_DOMO TRANSFERASESDM00367|P06133|187-461: G156-F377 UDP-glycosyltransferases signature:W271-Q314 MOTIFS 46 7511647CD1 91 S46 T43 T51 Signal_cleavage: M1-A20SPSCAN 2-AMINO-3-KETO-BUTYRATE-COA LIGASE EC BLAST_PRODOM 2.3.1.29LIGASE TRANSFERASE ACYLTRANSFERASE PD168670: M1-I30 47 7510335CD1 275S21 S221 S227 T61 N244 Signal Peptide: M6-G29 HMMER Acyl-CoAdehydrogenase, N-terminal domain: L94-A213 HMMER_PFAM Acyl-CoAdehydrogenases proteins BL00072: L139-E149, BLIMPS_BLOCKS Y241-P253Acyl-CoA dehydrogenases signatures: L216-S272 PROFILESCAN ACYLCOADEHYDROGENASE BLAST_PRODOM VERYLONGCHAIN SPECIFIC PRECURSOR VLCADOXIDOREDUCTASE FLAVOPROTEIN FAD FATTY PD015520: M1-V93 PROTEINDEHYDROGENASE ACYLCOA BLAST_PRODOM OXIDOREDUCTASE FLAVOPROTEIN FADOXIDASE FATTY ACID METABOLISM PD000396: V93-H256 ACYL-COA DEHYDROGENASESDM00853 BLAST_DOMO |P48818|85-478: D85-I250 |P45857|1-377: L94-I250|P26440|40-420: L94-I250 |P45867|3-379: L94-I250 Acyl-CoA dehydrogenasessignature 1: C215-S227 MOTIFS 48 7510337CD1 618 S21 S221 S227 N244 N365Signal Peptide: M6-G29 HMMER S588 T61 T351 T364 T545 Acyl-CoAdehydrogenase, C-terminal domain: G327-A473 HMMER_PFAM Acyl-CoAdehydrogenase, middle domain: C215-L323 HMMER_PFAM Acyl-CoAdehydrogenase, N-terminal domain: W133-A213 HMMER_PFAM Acyl-CoAdehydrogenases proteins BL00072: L139-E149, BLIMPS_BLOCKS Y241-G253,G290-F330, M344-E394, E432-L474 Acyl-CoA dehydrogenases signatures:L216-T272 PROFILESCAN Acyl-CoA dehydrogenases signatures: A415-I467PROFILESCAN DEHYDROGENASE ACYL COA VERY LONG BLAST_PRODOM CHAIN SPECIFICPRECURSOR VLCAD OXIDOREDUCTASE FLAVOPROTEIN FAD FATTY PD013349:L484-E609 PROTEIN DEHYDROGENASE ACYL COA BLAST_PRODOM OXIDOREDUCTASEFLAVOPROTEIN FAD OXIDASE FATTY ACID METABOLISM PD000396: V93-M404,V93-T307, L337-A473 ACYL COA DEHYDROGENASE VERY LONG BLAST_PRODOM CHAINSPECIFIC PRECURSOR VLCAD OXIDOREDUCTASE FLAVOPROTEIN FAD FATTY PD015520:M1-V93 ACYL-COA DEHYDROGENASES-DM00853 BLAST_DOMO |P48818|85-478:D85-M478 |P45857|1-377: L94-A473 |P45867|3-379: L94-A473 |Q06319|3-383:L136-I467 Acyl-CoA dehydrogenases signature 1: C215-S227 MOTIFS Acyl-CoAdehydrogenases signature 2: Q435-D454 MOTIFS 49 7510353CD1 454 S29 S34S46 S64 signal_cleavage: M1-C51 SPSCAN S326 T95 T177 T255 T356 Y117 Y259Signal Peptide: M1-A21 HMMER Polyprenyl synthetase: R110-Q417 HMMER_PFAMPolyprenyl synthetases proteins BL00723: G121-V131, BLIMPS_BLOCKSD169-C183, T255-M280 Polyprenyl synthetases signatures: A279-C368PROFILESCAN PYROPHOSPHATE SYNTHASE SYNTHETASE BLAST_PRODOM TRANSFERASEBIOSYNTHESIS ISOPRENE GERANYLTRANSTRANSFERASE DIPHOSPHATE GERANYLGERANYLFARNESYL PD000572: L111-I307, D344-D410 FARNESYL PYROPHOSPHATESYNTHETASE BLAST_PRODOM FPP FPS DIPHOSPHATE INCLUDES:DIMETHYLALLYLTRANSFERASE GERANYLTRANSTRANSFERASE TRANSFERASE PD122945:M67-R110 POLYPRENYL SYNTHETASES DM00371 BLAST_DOMO |P14324|7-267:S73-Q308, Q343-S369 |B34713|7-267: D74-Q308, Q343-S369 |P08524|2-264:K80-Q308, Q343-S369 |P49349|2-261: A77-Q308, Q343-S369 Polyprenylsynthetases signature 1: L166-G180 MOTIFS 50 7510470CD1 526 S249 S350S457 signal_cleavage: M1-A23 SPSCAN T71 T326 T372 T395 T500 T521Cytochrome P450: P42-K375, R397-A524 HMMER_PFAM Cytochrome P450 cysteineheme-iron ligand proteins BLIMPS_BLOCKS BL00086: H463-L494 CytochromeP450 cysteine heme-iron ligand PROFILESCAN signature: P443-Q495 P450superfamily signature PR00385: G314-A331, BLIMPS_PRINTS R332-L345,A367-E378, V464-C473 Mitochondrial P450 signature PR00408: W116-L131,BLIMPS_PRINTS L132-L142, F193-L211, G314-A331, R332-L345, T360-E378,Y446-I454, V464-C473, C473-L484 CYTOCHROME P450 ELECTRON TRANSPORTBLAST_PRODOM OXIDOREDUCTASE PRECURSOR MONOOXYGENASE MEMBRANE HEMESTEROID PD002412: M1-W49 CYTOCHROME P450 DM00022 BLAST_DOMO|P19099|84-494: G84-R374, T395-P518 |P15150|83-494: L83-R374, T395-P518|P30099|94-501: G84-R374, T395-P518 |P15538|84-494: G84-R374, T318-P518Cytochrome P450 cysteine heme-iron ligand MOTIFS signature: F466-G475 517504648CD1 527 S21 S221 S227 N244 N365 Signal Peptide: M6-G29 HMMER S488T61 T351 T364 Acyl-CoA dehydrogenase, C-terminal doma: G327-C477HMMER_PFAM Acyl-CoA dehydrogenase, middle domain: C215-L323 HMMER_PFAMAcyl-CoA dehydrogenase, N-terminal doma: L94-A213 HMMER_PFAM Acyl-CoAdehydrogenases proteins BL00072: L139-E149, BLIMPS_BLOCKS Y241-G253,G290-F330, M344-E394, E432-L474 Acyl-CoA dehydrogenases signatures:L216-T272, PROFILESCAN A415-I467 PROTEIN DEHYDROGENASE ACYL-COABLAST_PRODOM OXIDOREDUCTASE FLAVOPROTEIN FAD OXIDASE FATTY ACIDMETABOLISM: PD000396: V93-M404, L337-A473 ACYL-COA DEHYDROGENASE VERYLONG BLAST_PRODOM CHAIN SPECIFIC PRECURSOR VLCAD OXIDOREDUCTASEFLAVOPROTEIN FAD FATTY: PD015520: M1-V93 ACYL-COA DEHYDROGENASESBLAST_DOMO DM00853|P48818|85-478: D85-M478; DM00853|P45857|1-377:L94-A473; DM00853|P45867|3-379: L94-A473; DM00853|Q06319|3-383:L136-I467 Acyl-CoA dehydrogenases signature 1: C215-S227 MOTIFS Acyl-CoAdehydrogenases signature 2: Q435-D454 MOTIFS 52 7512747CD1 183 S84 S157T118 2-hydroxychromene-2-carboxylate isomer: T7-E169 HMMER_PFAM Y12ISOMERASE PROTEIN STRANSFERASE BLAST_PRODOM CHROMOSOME DIOXYGENASE2HYDROXYCHROMENE2-CARBOXYLATE PLASMID THE GLUTATHIONE MITOCHONDRIALPD008447: L26-G168 53 7510146CD1 329 S249 T71 T318 signal_cleavage:M1-A23 SPSCAN T326 Mitochondrial P450 signature PR00408: W116-L131,BLIMPS_PRINTS L132-L142, F193-L211 CYTOCHROME P450 ELECTRON TRANSPORTBLAST_PRODOM OXIDOREDUCTASE PRECURSOR MONOOXYGENASE MEMBRANE HEMESTEROID PD002412: M1-W49 CYTOCHROME P450 DM00022 BLAST_DOMO|P15538|84-494: G84-T318 |P19099|84-494: G84-T318 |P15150|83-494:L83-T318 |P30099|94-501: G84-T318

TABLE 4 Polynucleotide SEQ ID NO:/ Incyte ID/Sequence Length SequenceFragments 54/7499940CB1/ 1-1640, 9-1624, 57-659, 57-677, 57-752, 57-775,57-776, 57-834, 57-836, 57-843, 57-901, 57-951, 63-845, 591-1140, 1640614-1480, 630-1480, 637-1480, 655-1272, 666-1480, 667-1480, 670-1480,671-1480, 706-1250, 709-1479, 742-1480, 743-1480, 772-1480, 803-1480,824-1295, 831-1480, 847-1479, 868-1479, 870-1136, 883-1479, 885-1409,893-1586, 905-1097, 920-1479, 976-1432, 1013-1312, 1025-1470, 1026-1605,1077-1459, 1083-1453, 1131-1473, 1167-1453, 1221-1498, 1228-1625,1280-1587, 1280-1596, 1291-1572, 1423-1614, 1451-1638, 1494-1614,1495-1622, 1557-1640 55/3329870CB1/ 1-311, 20-768, 73-729, 432-954,477-640, 563-1244, 672-1323, 747-1291, 766-1026, 892-1193, 892-1326,918-1521, 2373 1094-1751, 1113-1748, 1162-1845, 1165-1721, 1175-1777,1348-1907, 1498-2069, 1725-2373, 1767-2010, 1834-2287, 1837-2116,1987-2293, 2001-2259, 2004-2288 56/7500698CB1/600 1-171, 2-134, 2-172,2-600, 3-172, 9-131, 9-169, 10-172, 11-134, 15-172, 16-168, 114-387,114-391, 122-387, 170-226, 186-375, 186-430, 207-528, 213-459, 214-478,216-480, 221-543, 234-475, 234-554, 250-482, 260-531, 262-600, 265-537,271-582, 290-543, 295-466, 297-546, 299-534, 300-554, 301-559, 302-569,313-596, 325-534, 342-600, 386-568, 438-579, 522-552 57/7500223CB1/1-136, 1-263, 1-1566, 3-255, 7-150, 8-253, 9-192, 10-277, 10-308,14-272, 23-273, 31-299, 31-442, 32-174, 33-263, 1579 36-242, 36-364,37-291, 40-177, 42-306, 42-311, 51-313, 51-385, 63-308, 64-384, 65-320,72-363, 74-192, 79-317, 84-264, 89-344, 91-284, 91-369, 92-348, 101-388,103-365, 109-340, 111-437, 112-393, 118-400, 138-425, 139-727, 207-666,208-342, 256-407, 259-568, 265-356, 294-564, 302-896, 325-821, 340-483,342-684, 356-941, 372-1078, 389-645, 403-861, 412-653, 412-922, 419-726,435-594, 435-685, 435-728, 435-892, 435-1099, 435-1115, 435-1220,435-1223, 436-652, 436-714, 436-917, 438-1078, 445-671, 453-766,454-672, 454-1112, 459-747, 462-675, 465-634, 468-747, 471-678, 471-775,471-944, 476-1333, 478-729, 478-977, 481-663, 481-1108, 482-710,496-708, 497-849, 497-1003, 498-893, 504-1078, 521-1076, 527-791,531-747, 533-773, 533-774, 538-748, 540-657, 548-1166, 553-780, 555-662,555-969, 567-1026, 568-994, 572-812, 581-1057, 585-800, 588-848,588-1269, 592-1144, 592-1270, 598-942, 602-1034, 603-848, 603-865,603-868, 605-1103, 613-719, 614-1250, 615-1175, 621-851, 621-881,628-962, 631-865, 636-874, 636-1272, 637-893, 643-906, 643-1047,646-904, 652-883, 652-905, 652-1039, 652-1041, 662-946, 666-1370,673-895, 673-1442, 682-1111, 687-1016, 687-1225, 690-1202, 696-916,701-968, 704-1068, 707-1119, 708-876, 708-962, 710-1275, 712-955,712-997, 722-1274, 724-1313, 727-959, 730-978, 732-1284, 733-975,734-982, 737-1242, 739-931, 739-978, 742-987, 743-984, 743-1011,743-1341, 743-1449, 745-1019, 746-1269, 748-1050, 749-1115, 753-1001,760-1291, 763-1022, 767-1082, 767-1329, 768-993, 773-1202, 773-1206,774-1252, 776-1201, 781-1346, 785-1223, 788-1286, 789-1039, 794-1035,800-1039, 802-1326, 805-996, 805-1269, 811-1349, 815-1263, 819-1027,819-1080, 819-1115, 826-1269, 829-1076, 831-917, 838-1087, 838-1108,845-1230, 847-1118, 847-1263, 848-1109, 849-1127, 850-1269, 853-1374,861-1099, 861-1101, 864-1103, 867-1269, 868-1141, 868-1147, 868-1173,871-1161, 872-1270, 872-1459, 877-1135, 886-1257, 887-1149, 892-1565,901-1142, 903-1520, 906-1171, 917-1144, 919-1439, 920-1530, 922-1474,926-1184, 932-1557, 938-1117, 940-1191, 944-1251, 946-1577, 949-1565,951-1176, 953-1269, 954-1198, 958-1198, 965-1175, 965-1338, 966-1497,968-1558, 969-1564, 973-1457, 974-1558, 978-1209, 978-1210, 978-1211,979-1237, 979-1268, 979-1555, 980-1262, 980-1579, 981-1565, 983-1229,988-1271, 993-1222, 993-1227, 993-1362, 993-1489, 996-1113, 1005-1227,1007-1249, 1007-1262, 1007-1277, 1010-1575, 1012-1216, 1017-1362,1018-1561, 1019-1322, 1021-1527, 1023-1251, 1026-1292, 1031-1579,1034-1347, 1038-1557, 1041-1276, 1046-1293, 1046-1310, 1048-1579,1051-1273, 1053-1270, 1068-1350, 1071-1577, 1072-1579, 1074-1260,1074-1337, 1084-1565, 1086-1565, 1088-1379, 1089-1285, 1089-1569,1092-1579, 1101-1539, 1101-1574, 1104-1569, 1107-1568, 1108-1560,1109-1555, 1109-1579, 1115-1568, 1126-1569, 1133-1565, 1133-1570,1134-1564, 1134-1579, 1136-1565, 1136-1571, 1136-1579, 1140-1565,1144-1572, 1146-1566, 1150-1565, 1159-1249, 1164-1385, 1165-1482,1167-1436, 1172-1565, 1174-1566, 1176-1249, 1177-1476, 1181-1249,1189-1566, 1199-1565, 1200-1565, 1205-1499, 1209-1565, 1212-1557,1214-1488, 1215-1572, 1215-1579, 1216-1565, 1221-1568, 1225-1551,1229-1457, 1229-1556, 1233-1565, 1235-1568, 1238-1496, 1243-1567,1252-1491, 1253-1557, 1256-1520, 1257-1528, 1260-1566, 1261-1571,1263-1558, 1263-1559, 1263-1565, 1267-1568, 1280-1567, 1286-1561,1298-1579, 1300-1579, 1308-1579, 1310-1565, 1310-1569, 1315-1565,1319-1565, 1320-1566, 1321-1579, 1323-1536, 1323-1565, 1361-1574,1366-1543, 1366-1565, 1368-1579, 1381-1579, 1382-1565, 1389-1565,1396-1572, 1399-1521, 1409-1530, 1417-1567, 1440-1563, 1440-1579,1483-1579, 1508-1568 58/7500295CB1/ 1-264, 1-1567, 2-137, 4-256, 8-151,9-254, 10-193, 11-278, 11-309, 15-273, 24-274, 32-300, 32-443, 33-175,34-264, 1601 37-243, 37-365, 38-292, 41-178, 43-307, 43-312, 52-314,52-386, 53-438, 59-438, 64-309, 65-385, 66-321, 73-264, 75-193, 80-318,85-265, 90-345, 92-285, 92-370, 93-349, 102-389, 104-366, 110-341,112-438, 113-394, 119-401, 139-426, 140-728, 208-667, 209-343, 209-438,257-408, 260-569, 266-357, 295-565, 303-897, 326-820, 341-484, 343-685,357-942, 373-1079, 390-646, 404-862, 413-654, 413-923, 420-727, 436-595,436-686, 436-729, 436-893, 436-1100, 436-1116, 436-1221, 436-1224,437-653, 437-670, 437-715, 437-918, 439-1079, 440-482, 446-672, 454-767,455-673, 455-1113, 460-748, 463-676, 465-627, 466-635, 469-748, 472-679,472-776, 472-945, 477-1334, 479-730, 479-978, 482-664, 482-1109,483-711, 497-709, 498-850, 498-1004, 499-894, 505-1079, 522-1077,528-792, 532-748, 534-774, 534-775, 539-749, 541-658, 549-1167, 554-781,556-663, 556-970, 568-1027, 569-995, 573-809, 582-1058, 586-801,589-849, 589-1270, 593-1145, 593-1271, 599-943, 603-1035, 604-849,604-866, 604-869, 606-1104, 608-732, 614-720, 615-1251, 616-1176,621-886, 622-852, 622-882, 629-963, 632-866, 637-875, 637-1273, 638-894,644-907, 644-1048, 647-905, 653-884, 653-906, 653-1040, 653-1042,663-947, 667-1371, 674-896, 674-1443, 683-1112, 688-1017, 688-1226,691-1203, 697-917, 702-969, 705-1069, 708-1120, 709-877, 709-963,711-1276, 713-956, 713-998, 723-1275, 725-1314, 727-1003, 728-960,731-979, 733-1285, 734-976, 735-983, 738-1243, 740-932, 740-979,743-988, 744-984, 744-985, 744-1012, 744-1342, 744-1450, 746-1020,747-1270, 749-1051, 750-1116, 754-1002, 761-1292, 764-1023, 768-1083,768-1330, 769-994, 774-1203, 774-1207, 775-1253, 777-1202, 782-1347,786-1224, 789-1287, 790-1040, 795-1036, 803-1040, 803-1327, 806-997,808-1270, 812-935, 812-1350, 812-1525, 816-1264, 819-981, 820-1028,820-1081, 820-1116, 827-1270, 830-1077, 832-918, 839-1088, 839-11091109, 845-1476, 846-1231, 848-1119, 848-1264, 849-1110, 850-1128,851-1270, 852-1070, 854-1375, 862-1100, 862-1102, 865-1104, 868-1270,869-1142, 869-1148, 869-1174, 872-1162, 873-1271, 873-1460, 878-1136,887-1258, 888-1150, 893-1566, 902-1143, 904-1521, 907-1172, 918-1145,919-1211, 920-1440, 921-1531, 923-1475, 927-1185, 933-1558, 939-1118,941-1192, 945-1252, 947-1578, 950-1566, 952-1177, 954-1270, 955-1199,955-1205, 959-1199, 966-1176, 966-1339, 967-1498, 969-1559, 970-1565,974-1458, 975-1559, 979-1210, 979-1211, 979-1212, 980-1238, 980-1269,980-1556, 981-1263, 981-1580, 982-1566, 984-1230, 989-1272, 994-1223,994-1228, 994-1363, 994-1490, 997-1114, 998-1547, 1006-1204, 1006-1228,1008-1250, 1008-1263, 1008-1278, 1011-1576, 1013-1217, 1018-1363,1019-1562, 1020-1323, 1022-1528, 1023-1312, 1024-1252, 1027-1293,1028-1583, 1032-1588, 1035-1348, 1038-1523, 1039-1558, 1039-1599,1042-1277, 1045-1307, 1047-1294, 1047-1311, 1049-1581, 1052-1274,1054-1271, 1065-1403, 1069-1351, 1072-1578, 1073-1584, 1075-1261,1075-1338, 1085-1566, 1086-1586, 1087-1566, 1089-1279, 1089-1380,1090-1286, 1090-1570, 1093-1581, 1095-1250, 1101-1572, 1102-1540,1102-1594, 1103-1347, 1105-1570, 1106-1390, 1108-1569, 1109-1561,1110-1556, 1110-1584, 1114-1365, 1114-1377, 1116-1566, 1116-1569,1116-1584, 1117-1565, 1118-1421, 1123-1566, 1127-1570, 1129-1570,1134-1566, 1134-1571, 1135-1250, 1135-1565, 1135-1601, 1136-1566,1137-1566, 1137-1572, 1137-1580, 1139-1566, 1140-1404, 1141-1566,1143-1585, 1145-1573, 1146-1566, 1147-1421, 1147-1423, 1147-1567,1148-1556, 1151-1566, 1153-1250, 1154-1250, 1156-1566, 1158-1423,1160-1250, 1165-1386, 1165-1418, 1166-1483, 1168-1357, 1168-1437,1168-1566, 1170-1436, 1172-1438, 1173-1566, 1175-1567, 1177-1250,1178-1446, 1178-1477, 1182-1250, 1182-1418, 1188-1567, 1190-1567,1191-1417, 1200-1250, 1200-1566, 1201-1566, 1202-1566, 1206-1500,1210-1554, 1210-1566, 1213-1558, 1214-1566, 1215-1471, 1215-1489,1215-1566, 1216-1573, 1216-1585, 1217-1566, 1218-1596, 1222-1569,1226-1552, 1230-1458, 1230-1557, 1233-1566, 1234-1566, 1235-1566,1236-1569, 1239-1497, 1244-1568, 1254-1558, 1257-1521, 1258-1529,1261-1567, 1262-1572, 1264-1510, 1264-1559, 1264-1560, 1264-1566,1268-1569, 1281-1568, 1287-1562, 1287-1568, 1296-1592, 1299-1595,1301-1566, 1301-1599, 1309-1580, 1311-1566, 1311-1570, 1313-1574,1316-1566, 1320-1566, 1321-1567, 1324-1537, 1324-1566, 1327-1558,1361-1598, 1362-1589, 1367-1544, 1367-1566, 1369-1580, 1382-1599,1383-1566, 1390-1566, 1390-1592, 1397-1573, 1400-1522, 1410-1531,1418-1568, 1421-1566, 1441-1564, 1441-1599, 1484-1597, 1509-156959/7502095CB1/ 1-173, 1-190, 1-1427, 30-190, 47-395, 66-218, 83-173,123-190, 124-190, 174-244, 174-269, 176-874, 192-497, 213-471, 1433214-745, 250-916, 334-629, 385-657, 479-545, 514-1260, 533-1068,550-839, 553-837, 573-1137, 625-1152, 927-1433, 1284-1425, 1284-1433,1317-1433 60/7500507CB1/ 1-250, 1-286, 1-1894, 100-709, 100-798,102-924, 110-389, 112-405, 112-529, 411-1232, 425-994, 430-1014,439-636, 1919 440-896, 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1277-1447,1277-1556, 1280-1497, 1280-1558, 1281-1533, 1281-1550, 1290-1478,1290-1551, 1290-1562, 1297-1522, 1300-1558, 1301-1485, 1302-1559,1304-1858, 1311-1549, 1313-1558, 1317-1561, 1320-1558, 1321-1827,1328-1540, 1334-1614, 1334-1787, 1335-1533, 1339-1531, 1339-1608,1341-1485, 1342-1870, 1347-1618, 1347-1622, 1349-1570, 1391-1472,1408-1669, 1429-1598, 1434-1604, 1445-1741, 1445-1752, 1451-1717,1454-1535, 1533-1829, 1544-2160, 1557-1839, 1557-1877, 1557-2084,1557-2087, 1557-2101, 1557-2160, 1558-2107, 1559-1699, 1559-2119,1559-2143, 1559-2155, 1561-1786, 1561-1805, 1562-2032, 1562-2087,1565-1827, 1565-1857, 1568-1782, 1568-1794, 1570-1834, 1571-2149,1571-2160, 1572-1847, 1572-2120, 1573-1836, 1573-2123, 1574-1830,1574-2153, 1575-1853, 1575-1867, 1575-1941, 1578-2152, 1579-1784,1579-1837, 1579-1850, 1579-2157, 1580-1787, 1580-1885, 1584-2039,1585-1858, 1585-1934, 1586-1857, 1586-1870, 1589-1812, 1589-2160,1596-2160, 1597-1874, 1598-1805, 1598-1868, 1598-1899, 1598-2109,1598-2160, 1601-2159, 1602-2160, 1604-1892, 1604-2054, 1604-2157,1607-1892, 1607-2120, 1609-1882, 1614-1840, 1614-1871, 1614-1873,1614-1885, 1614-2160, 1617-1854, 1617-1906, 1618-2136, 1620-1874,1622-1859, 1622-1913, 1622-2160, 1624-1868, 1624-2160, 1625-2160,1627-1900, 1629-2157, 1630-1914, 1632-2111, 1634-2160, 1635-2160,1637-1871, 1638-2152, 1639-1891, 1641-2154, 1643-1921, 1645-1841,1645-2016, 1646-2160, 1647-1940, 1647-2044, 1648-2148, 1649-1904,1651-1938, 1651-1939, 1655-1883, 1658-2111, 1659-2150, 1662-2160,1679-2160, 1680-2160, 1681-1949, 1681-1973, 1688-1936, 1689-1942,1689-1958, 1689-1962, 1689-1963, 1689-1964, 1689-1965, 1690-2160,1691-2074, 1691-2132, 1692-2160, 1693-1928, 1693-1954, 1693-2139,1694-1953, 1694-2003, 1694-2090, 1694-2160, 1696-1926, 1696-1965,1696-2012, 1696-2160, 1700-1931, 1700-1934, 1700-1954, 1700-1971,1700-2023, 1701-2160, 1702-1858, 1702-1998, 1703-1949, 1703-1985,1705-2160, 1706-1946, 1706-1995, 1707-1950, 1707-1970, 1707-2160,1711-2065, 1712-1985, 1713-2160, 1717-1996, 1720-2160, 1723-2160,1733-2032, 1733-2160, 1734-2160, 1735-1979, 1735-2160, 1736-2160,1737-1985, 1737-2016, 1737-2160, 1739-2024, 1740-2160, 1742-1991,1742-2160, 1743-1984, 1745-1991, 1745-2015, 1745-2160, 1749-2160,1750-2160, 1751-2160, 1752-1987, 1754-1879, 1754-2159, 1754-2160,1755-2159, 1756-2160, 1757-2159, 1757-2160, 1758-2019, 1758-2160,1760-2159, 1760-2160, 1761-2160, 1762-2010, 1762-2160, 1763-2151,1763-2159, 1763-2160, 1767-2160, 1769-2019, 1770-2023, 1771-2007,1771-2160, 1774-2159, 1774-2160, 1775-2036, 1775-2144, 1775-2160,1776-2160, 1777-2092, 1780-2113, 1780-2160, 1781-2160, 1782-2160,1783-2160, 1784-2160, 1785-2157, 1791-2160, 1792-2160, 1793-2135,1794-2115, 1794-2119, 1795-2160, 1796-2152, 1797-2160, 1798-2045,1798-2058, 1798-2160, 1801-2159, 1801-2160, 1802-2160, 1805-2160,1806-2082, 1806-2160, 1807-2079, 1808-2132, 1809-2157, 1809-2159,1809-2160, 1812-2096, 1815-2014, 1815-2021, 1822-2108, 1823-2117,1823-2160, 1829-2160, 1831-2160, 1838-2097, 1838-2160, 1840-2102,1840-2159, 1840-2160, 1845-2160, 1846-2160, 1849-2058, 1849-2147,1851-2160, 1853-2160, 1855-2160, 1856-2130, 1856-2160, 1860-2152,1860-2160, 1862-2160, 1865-2067, 1866-2159, 1872-2160, 1873-2160,1874-2098, 1874-2144, 1874-2160, 1876-2160, 1877-2160, 1879-1989,1879-2082, 1879-2117, 1879-2160, 1880-2105, 1880-2160, 1881-2160,1882-2160, 1883-2160, 1884-2160, 1885-2160, 1887-2160, 1888-2160,1892-2085, 1892-2102, 1892-2142, 1894-2160, 1900-2160, 1902-2160,1903-2119, 1903-2160, 1905-2160, 1906-2160, 1908-2160, 1910-2108,1910-2125, 1910-2160, 1911-2160, 1913-2160, 1917-2160, 1919-2140,1919-2160, 1920-2160, 1921-2160, 1922-2159, 1922-2160, 1923-2160,1924-2160, 1925-2112, 1925-2132, 1925-2160, 1927-2160, 1928-2160,1931-2160, 1933-2100, 1933-2160, 1940-2120, 1940-2160, 1945-2157,1945-2160, 1946-2160, 1950-2160, 1951-2138, 1951-2160, 1952-2158,1952-2160, 1953-2160, 1954-2160, 1956-2146, 1956-2160, 1959-2160,1960-2160, 1961-2102, 1964-2160, 1966-2160, 1971-2160, 1972-2160,1977-2160, 1979-2097, 1979-2106, 1985-2160, 1986-2160, 1997-2160,1999-2160, 2000-2147, 2000-2157, 2003-2160, 2005-2160, 2006-2160,2015-2160, 2018-2124, 2018-2160, 2021-2160, 2035-2160, 2036-2085,2036-2160, 2039-2160, 2040-2160, 2049-2160, 2054-2160, 2070-2160,2075-2160, 2076-2160, 2078-2160, 2093-2160, 2095-2160 105/7512747CB1/1-903, 25-198, 45-143, 62-186, 146-431, 220-654, 220-903, 236-645,241-894, 246-437, 249-737, 254-796, 261-854 903 264-903, 268-814,268-823, 268-864, 268-883, 269-815, 269-816, 269-830, 272-861, 282-836,291-900, 292-895, 305-823, 307-436, 317-550, 319-858, 321-852, 331-589,331-591, 331-891, 332-792, 332-901, 346-671, 350-894, 358-821, 358-903,359-898, 361-603, 362-903, 365-894, 383-879, 385-621, 386-624, 392-881,393-903, 396-902, 403-881, 405-876, 407-841, 407-894, 408-903, 409-851,416-690, 416-903, 418-894, 421-648, 424-881, 424-894, 425-895, 427-892,428-858, 429-646, 429-889, 429-894, 430-903, 431-659, 431-771, 431-894,431-895, 431-901, 431-902, 431-903, 432-894, 434-694, 434-771, 434-880,434-903, 436-894, 438-903, 439-685, 439-697, 440-890, 440-894, 441-894,443-895, 444-630, 445-892, 445-903, 446-730, 446-734, 446-894, 447-879,448-894, 449-894, 450-903, 452-678, 453-894, 453-903, 455-894, 459-890,459-903, 461-894, 461-903, 463-903, 464-665, 464-894, 465-903, 470-894,471-894, 475-678, 475-886, 475-894, 476-790, 476-890, 476-894, 476-903,478-894, 479-894, 482-903, 483-894, 485-858, 485-894, 486-757, 486-894,487-894, 490-751, 490-894, 491-894, 493-650, 494-891, 497-894, 499-894,503-709, 507-895, 507-903, 508-903, 509-826, 510-894, 511-755, 511-890,512-790, 512-801, 512-890, 513-891, 514-894, 515-850, 515-903, 518-894,519-890, 519-903, 520-894, 525-766, 525-894, 529-894, 532-878, 532-893,532-895, 533-856, 538-736, 539-722, 539-772, 542-772, 544-894, 547-766,551-805, 551-894, 553-855, 555-800, 555-894, 556-609, 556-779, 561-903,563-890, 563-892, 564-890, 565-890, 566-894, 569-864, 571-816, 574-892,574-894, 575-842, 577-861, 577-894, 579-890, 580-893, 580-894, 581-895,582-865, 583-890, 583-894, 584-894, 585-894, 588-837, 600-894, 602-890,602-893, 602-894, 607-894, 608-895, 610-890, 610-894, 610-903, 614-890,618-890, 619-869, 619-893, 619-900, 620-833, 623-894, 624-888, 625-890,625-891, 631-903, 633-894, 634-894, 634-901, 635-826, 635-861, 635-863,635-890, 637-829, 637-889, 639-892, 646-890, 647-890, 648-890, 649-903,650-894, 660-903, 662-891, 663-887, 663-900, 664-894, 665-894, 665-895,669-892, 675-903, 679-903, 682-903, 691-854, 701-894, 701-903, 702-895,708-903, 710-894, 735-903, 753-890, 753-891, 753-894, 762-903, 765-890,767-895, 780-903, 782-903, 789-901, 800-894, 827-903 106/7510146CB1/1-233, 2-184, 8-582, 8-625, 8-658, 8-665, 8-753, 8-2510, 13-225, 21-816,144-617, 144-821, 144-894, 191-788, 191-840, 2510 261-561, 261-692,261-695, 261-703, 261-734, 261-788, 261-810, 261-865, 261-903, 261-920,266-843, 271-983, 304-595, 304-827, 304-914, 309-860, 311-695, 341-1042,344-1048, 369-801, 376-849, 407-694, 409-941, 409-1024, 436-1024,442-982, 443-1064, 469-1097, 471-1150, 513-881, 526-1167, 533-1144,548-881, 554-1179, 591-1356, 605-900, 931-1637, 980-1676, 1078-1650,1083-1605, 1103-1592, 1113-1679, 1129-1830, 1131-1738, 1160-1586,1176-1808, 1183-1732, 1183-1823, 1202-1779, 1225-1817, 1231-1823,1242-1712, 1280-1844, 1290-1835, 1290-1897, 1301-1839, 1367-1636,1384-1628

TABLE 5 Polynucleotide SEQ ID NO: Incyte Project ID: RepresentativeLibrary 54 7499940CB1 MONOTXN05 55 3329870CB1 SEMVNOT03 56 7500698CB1BRAFTUE03 57 7500223CB1 LUNGNOT02 58 7500295CB1 LUNGNOT02 59 7502095CB1MLP000028 60 7500507CB1 BMARNOT03 61 7500840CB1 PGANNOT03 62 7493620CB1ADMEDNV17 63 7494697CB1 HELAUNT01 64 8146738CB1 LUNGNOT34 65 7500114CB1OVARDIR01 66 7500197CB1 LUNGTUT07 67 7500145CB1 FIBRUNT02 68 7500874CB1FIBRUNT02 69 7500495CB1 SINTFET03 70 7500194CB1 BRAITDR03 71 7500871CB1FIBRUNT02 72 7500873CB1 FIBRUNT02 73 7503491CB1 UTREDIT07 74 7503427CB1FIBPFEN06 75 7503547CB1 BRABDIE02 76 1932641CB1 COLNNOT16 77 6892447CB1ARTANOT06 78 7503416CB1 EPIPUNA01 79 7503874CB1 OVARTUE01 80 7503454CB1BRSTNOT16 81 7503528CB1 NGANNOT01 82 7503705CB1 HEAONOE01 83 7503707CB1HEAONOE01 85 70819231CB1  THYRNOT03 86 7504066CB1 HELAUNT01 8790001862CB1  COLENOR03 88 7503046CB1 SINTFEE01 89 7503211CB1 KIDNNOC0190 7503264CB1 ISLTNOT01 93 7503199CB1 TESTNOT03 94 7511530CB1 ADRENOT0395 7511535CB1 ENDANOT01 96 7511536CB1 ENDANOT01 97 7511583CB1 SCORNOT0498 7511395CB1 LIVRDIT02 99 7511647CB1 BRAINOT11 100 7510335CB1 SINTNOR01101 7510337CB1 SINTNOR01 102 7510353CB1 UCMCNOT02 103 7510470CB1KIDNNOC01 104 7504648CB1 SINTNOR01 105 7512747CB1 KIDNNOT34 1067510146CB1 KIDNNOC01

TABLE 6 Library Vector Library Description ADMEDNV17 PCR2-TOPOTA Librarywas constructed using pooled cDNA from different donors. cDNA wasgenerated using mRNA isolated from pooled skeletal muscle tissue removedfrom ten 21 to 57-year-old Caucasian male and female donors who diedfrom sudden death; from pooled thymus tissue removed from nine 18 to32-year-old Caucasian male and female donors who died from sudden death;from pooled liver tissue removed from 32 Caucasian male and femalefetuses who died at 18-24 weeks gestation due to spontaneous abortion;from kidney tissue removed from 59 Caucasian male and female fetuses whodied at 20-33 weeks gestation due to spontaneous abortion; and frombrain tissue removed from a Caucasian male fetus who died at 23 weeksgestation due to fetal demise. ADRENOT03 PSPORT1 Library was constructedusing RNA isolated from the adrenal tissue of a 17-year-old Caucasianmale, who died from cerebral anoxia. ARTANOT06 pINCY Library wasconstructed using RNA isolated from aortic adventitia tissue removedfrom a 48-year-old Caucasian male. BMARNOT03 pINCY Library wasconstructed using RNA isolated from the left tibial bone marrow tissueof a 16-year-old Caucasian male during a partial left tibial ostectomywith free skin graft. Patient history included an abnormality of the redblood cells. Previous surgeries included bone and bone marrow biopsy,and soft tissue excision. Family history included osteoarthritis.BRABDIE02 pINCY This 5′ biased random primed library was constructedusing RNA isolated from diseased cerebellum tissue removed from thebrain of a 57-year-old Caucasian male who died from a cerebrovascularaccident. Serologies were negative. Patient history includedHuntington's disease, emphysema, and tobacco abuse (3-4 packs per day,for 40 years). BRAFTUE03 PCDNA2.1 This 5′ biased random primed librarywas constructed using RNA isolated from brain tumor tissue removed fromthe left frontal lobe of a 40-year-old Caucasian female during excisionof a cerebral meningeal lesion. Pathology indicated grade 4 gemistocyticastrocytoma. The patient presented with coma, epilepsy, and incontinenceof urine and stool, type II diabetes, abulia, and paralysis. Patienthistory included chronic nephritis and cesarean delivery. Patientmedications included Decadron and phenytoin sodium. BRAINOT11 pINCYLibrary was constructed using RNA isolated from brain tissue removedfrom the right temporal lobe of a 5-year-old Caucasian male during ahemispherectomy. Pathology indicated extensive polymicrogyria and mildto moderate gliosis (predominantly subpial and subcortical), consistentwith chronic seizure disorder. Family history included a cervicalneoplasm. BRAITDR03 PCDNA2.1 This random primed library was constructedusing RNA isolated from allocortex, cingulate posterior tissue removedfrom a 55-year-old Caucasian female who died from cholangiocarcinoma.Pathology indicated mild meningeal fibrosis predominately over theconvexities, scattered axonal spheroids in the white matter of thecingulate cortex and the thalamus, and a few scattered neurofibrillarytangles in the entorhinal cortex and the periaqueductal gray region.Pathology for the associated tumor tissue indicated well-differentiatedcholangiocarcinoma of the liver with residual or relapsed tumor. Patienthistory included cholangiocarcinoma, post-operative Budd-Chiarisyndrome, biliary ascites, hydrothorax, dehydration, malnutrition,oliguria and acute renal failure. Previous surgeries includedcholecystectomy and resection of 85% of the liver. BRSTNOT16 pINCYLibrary was constructed using RNA isolated from diseased breast tissueremoved from a 59-year-old Caucasian female during a unilateral extendedsimple mastectomy. Pathology for the associated tumor tissue indicatedan invasive lobular carcinoma with extension into ducts. Patient historyincluded liver cirrhosis, esophageal ulcer, hyperlipidemia, andneuropathy. COLENOR03 PCDNA2.1 Library was constructed using RNAisolated from colon epithelium tissue removed from a 13-year-oldCaucasian female who died from a motor vehicle accident. COLNNOT16 pINCYLibrary was constructed using RNA isolated from sigmoid colon tissueremoved from a 62-year-old Caucasian male during a sigmoidectomy andpermanent colostomy. ENDANOT01 PBLUESCRIPT Library was constructed usingRNA isolated from aortic endothelial cell tissue from an explanted heartremoved from a male during a heart transplant. EPIPUNA01 PSPORT1 Librarywas constructed using RNA isolated from untreated prostatic epithelialcell tissue removed from a 17-year-old Hispanic male. Serologies werenegative. FIBPFEN06 pINCY The normalized prostate stromal fibroblasttissue libraries were constructed from 1.56 million independent clonesfrom a prostate fibroblast library. Starting RNA was made fromfibroblasts of prostate stroma removed from a male fetus, who died after26 weeks' gestation. The libraries were normalized in two rounds usingconditions adapted from Soares et al., PNAS (1994) 91: 9228 and Bonaldoet al., Genome Research (1996) 6: 791, except that a significantlylonger (48-hours/round)reannealing hybridization was used. The librarywas then linearized and recircularized to select for insert containingclones as follows: plasmid DNA was prepped from approximately 1 millionclones from the normalized prostate stromal fibroblast tissue librariesfollowing soft agar transformation. FIBRUNT02 pINCY Library wasconstructed using RNA isolated from an untreated MG-63 cell line derivedfrom an osteosarcoma removed from a 14-year-old Caucasian male.HEAONOE01 PCDNA2.1 This 5′ biased random primed library was constructedusing RNA isolated from the aorta of a 39-year-old Caucasian male, Whodied from a gunshot wound. Serology was positive for cytomegalovirus(CMV). Patient history included tobacco abuse (one pack of cigarettesper day for 25 years), and occasionally cocaine, marijuna, and alcoholuse. HELAUNT01 pINCY Library was constructed using RNA isolated fromHeLa cells. The HeLa cell line is derived from cervical adenocarcinomaremoved from a 31-year-old Black female. ISLTNOT01 pINCY Library wasconstructed using RNA isolated from a pooled collection of pancreaticislet cells. KIDNNOC01 pINCY This large size-fractionated library wasconstructed using RNA isolated from pooled left and right kidney tissueremoved from a Caucasian male fetus, who died from Patau's syndrome(trisomy 13) at 20-weeks' gestation. KIDNNOT34 pINCY Library wasconstructed using RNA isolated from left kidney tissue obtained from an8-year-old Caucasian male who died from an intracranial hemorrhage. Thepatient was not taking any medications. LIVRDIT02 pINCY Library wasconstructed using RNA isolated from diseased liver tissue removed from a63-year-old Caucasian female during a liver transplant. Patient historyincluded primary biliary cirrhosis. LUNGNOT02 PBLUESCRIPT Library wasconstructed using RNA isolated from the lung tissue of a 47-year-oldCaucasian male, who died of a subarachnoid hemorrhage. LUNGNOT34 pINCYLibrary was constructed using RNA isolated from lung tissue removed froma 12-year-old Caucasian male. LUNGTUT07 pINCY Library was constructedusing RNA isolated from lung tumor tissue removed from the upper lobe ofa 50-year-old Caucasian male during segmental lung resection. Pathologyindicated an invasive grade 4 squamous cell adenocarcinoma. Patienthistory included tobacco use. Family history included skin cancer.MLP000028 PCR2-TOPOTA Library was constructed using pooled cDNA fromdifferent donors. cDNA was generated using mRNA isolated from thefollowing: aorta, cerebellum, lymph nodes, muscle, tonsil (lymphoidhyperplasia), bladder tumor (invasive grade 3 transitional cellcarcinoma.), breast (proliferative fibrocystic changes without atypiacharacterized by epithelial ductal hyperplasia, testicle tumor(embryonal carcinoma), spleen, ovary, parathyroid, ileum, breast skin,sigmoid colon, penis tumor (fungating invasive grade 4 squamous cellcarcinoma), fetal lung,, breast, fetal small intestine, fetal liver,fetal pancreas, fetal lung, fetal skin, fetal penis, fetal bone, fetalribs, frontal brain tumor (grade 4 gemistocytic astrocytoma), ovary(stromal hyperthecosis), bladder, bladder tumor (invasive grade 3transitional cell carcinoma), stomach, lymph node tumor (metastaticbasaloid squamous cell carcinoma), tonsil (reactive lymphoidhyperplasia), periosteum from the tibia, fetal brain, fetal spleen,uterus tumor, endometrial (grade 3 adenosquamous carcinoma), seminalvesicle, liver, aorta, adrenal gland, lymph node (metastatic grade 3squamous cell carcinoma), glossal muscle, esophagus, esophagus tumor(invasive grade 3 adenocarcinoma), ileum, pancreas, soft tissue tumorfrom the skull (grade 3 ependymoma), transverse colon, (benign familialpolyposis), rectum tumor (grade 3 colonic adenocarcinoma), rib tumor,(metastatic grade 3 osteosarcoma), lung, heart, placenta, thymus,stomach, spleen (splenomegaly with congestion), uterus, cervix (mildchronic cervicitis with focal squamous metaplasia), spleen tumor(malignant lymphoma, diffuse large cell type B-cell phenotype withabundant reactive T-cells and marked granulomatous response), umbilicalcord blood mononuclear cells, upper lobe lung tumor, (grade 3 squamouscell carcinoma), endometrium (secretory phase), liver, liver tumor(metastatic grade 2 neuroendocrine carcinoma), colon, umbilical cordblood, Th1 cells, nonactivated, umbilical cord blood, Th2 cells,nonactivated, coronary artery endothelial cells (untreated), coronaryartery smooth muscle cells, (untreated), coronary artery smooth musclecells (treated with TNF & IL-1 10 ng/ml each for 20 hours), bladder(mild chronic cystitis), epiglottis, breast skin, small intestine, fetalprostate stroma fibroblasts, prostate epithelial cells (PrEC cells),fetal adrenal glands, fetal liver, kidney transformed embryonal cellline (293-EBNA) (untreated), kidney transformed embryonal cell line(293-EBNA) (treated with 5Aza-2deoxycytidine for 72 hours), mammaryepithelial cells, (HMEC cells), peripheral blood monocytes (treated withIL-10 at time 0, 10 ng/ml, LPS was added at 1 hour at 5 ng/ml.Incubation 24 hours), peripheral blood monocytes (treated withanti-IL-10 at time 0, 10 ng/ml, LPS was added at 1 hour at 5 ng/ml.Incubation 24 hours), spinal cord, base of medulla (Huntington'schorea), thigh and arm muscle (ALS), breast skin fibroblast (untreated),breast skin fibroblast (treated with 9CIS Retinoic Acid 1 μM for 20hours), breast skin fibroblast (treated with TNF-alpha & IL-1 beta, 10ng/ml each for 20 hours), fetal liver mast cells, hematopoietic (Mastcells prepared from human fetal liver hematopoietic progenitor cells(CD34+ stem cells) cultured in the presence of hIL-6 and hSCF for 18days), epithelial layer of colon, bronchial epithelial cells (treatedfor 20 hours with 20% smoke conditioned media), lymph node, pooledperipheral blood mononuclear cells (untreated), pooled brain segments:striatum, globus pallidus and posterior putamen (Alzheimer's Disease),pituitary gland, umbilical cord blood, CD34+ derived dendritic cells(treated with SCF, GM-CSF & TNF alpha, 13 days), umbilical cord blood,CD34+ derived dendritic cells (treated with SCF, GM-CSF & TNF alpha, 13days followed by PMA/Ionomycin for 5 hours), small intestine, rectum,bone marrow neuroblastoma cell line (SH-SY5Y cells, treated with6-Hydroxydopamine 100 uM for 8 hours), bone marrow, neuroblastoma cellline (SH-SY5Y cells, untreated), brain segments from one donor:amygdala, entorhinal cortex, globus pallidus, substantia innominata,striatum, dorsal caudate nucleus, dorsal putamen, ventral nucleusaccumbens, archaecortex (hippocampus anterior and posterior), thalamus,nucleus raphe magnus, periaqueductal gray, midbrain, substantia nigra,and dentate nucleus, pineal gland (Alzheimer's Disease), preadipocytes(untreated), preadipocytes (treated with a peroxisomeproliferator-activated receptor gamma agonist, 1 microM, 4 hours),pooled prostate (adenofibromatous hyperplasia), pooled kidney, pooledadipocytes (untreated), pooled adipocytes (treated with human insulin),pooled mesentaric and abdomenal fat, pooled adrenal glands, pooledthyroid (normal and adenomatous hyperplasia), pooled spleen (normal andwith changes consistent with idiopathic thrombocytopenic purpura),pooled right and left breast, pooled lung, pooled nasal polyps, pooledfat, pooled synovium (normal and rhumatoid arthritis), pooled brain(meningioma, gemistocytic astrocytoma and Alzheimer's disease), pooledfetal colon, pooled colon: ascending, descending (chronic ulcerativecolitis), and rectal tumor (adenocarcinoma), pooled esophagus, normaland tumor (invasive grade 3 adenocarcinoma), pooled breast skinfibroblast (one treated w/9CIS Retinoic Acid and the other withTNF-alpha & IL-1 beta), pooled gallbladder (acute necrotizingcholecystitis with cholelithiasis (clinically hydrops), acutehemorrhagic cholecystitis with cholelithiasis, chronic cholecystitis andcholelithiasis), pooled fetal heart, (Patau's and fetal demise), pooledneurogenic tumor cell line, SK-N-MC, (neuroepitelioma, metastasis tosupra-orbital area, untreated) and neuron, NT-2 cell line, (treated withmouse leptin at 1 μg/ml and 9cis retinoic acid at 3.3 μM for 6 days),pooled ovary (normal and polycystic ovarian disease), pooled prostate,(adenofibromatous hyperplasia), pooled seminal vesicle, pooled smallintestine, pooled fetal small intestine, pooled stomach and fetalstomach, prostate epithelial cells, pooled testis (normal and embryonalcarcinoma), pooled uterus, pooled uterus tumor (grade 3 adenosquamouscarcinoma and leiomyoma), pooled uterus, endometrium, and myometrium,(normal and adenomatous hyperplasia with squamous metaplasia and focalatypia), pooled brain: (temporal lobe meningioma, cerebellum andhippocampus (Alzheimer's Disease), pooled skin, fetal lung, adrenaltumor (adrenal cortical carcinoma), prostate tumor (adenocarcinoma),fetal heart, fetal small intestine, ovary tumor (mucinous cystadenoma),ovary, ovary tumor (transitional cell carcinoma), disease prostate(adenofibromatous hyperplasia), fetal colon, uterus tumor (leiomyoma),temporal brain, submandibular gland, colon tumor (adenocarcinoma),ascending and transverse colon, ovary tumor (endometrioid carcinoma),lung tumor (squamous cell carcinoma), fetal brain, fetal lung, uretertumor (transitional cell carcinoma), untreated HNT cells, para-aorticsoft tissue, testis, seminal vesicle, diseased ovary (endometriosis),temporal lobe, myometrium, diseased gallbladder (cholecystitis,cholelithiasis), placenta, breast tumor (ductal adenocarcinoma), breast,lung tumor (liposarcoma), endometrium, abdominal fat, cervical spinedorsal root ganglion, thoracic spine dorsal root ganglion, diseasedthyroid (adenomatous hyperplasia), liver, kidney, fetal liver, NT-2cells (treated with mouse leptin and 9cis RA), K562 cells (treated with9cis RA), cerebellum, corpus callosum, hypothalamus, fetal brainastrocytes (treated with TNFa and IL-1b), inferior parietal cortex,posterior hippocampus, pons, thalamus, C3A cells (untreated), C3A cells(treated with 3-methylcholanthrene), testis, colon epithelial layer,pooled prostate, pooled liver, substantia nigra, thigh muscle, rib bone,fallopian tube tumor (endometrioid and serous adenocarcinoma), diseasedlung (idiopathic pulmonary disease), cingulate anterior allocortex andneocortex, cingulate posterior allocortex, auditory neocortex, frontalneocortex, orbital inferior neocortex, parietal superior neocortex,visual primary neocortex, dentate nucleus, posterior cingulate,cerebellum, vermis, inferior temporal cortex, medulla, posteriorparietal cortex, colon polyp, pooled breast, anterior and posteriorhippocampus, mesenteric and abdominal fat, pooled esophagus, pooledfetal kidney, pooled fetal liver, ileum, small intestine, pooledgallbladder, frontal and superior temporal cortex, pooled ovary, pooledendometrium, pooled prostate, pooled kidney, fetal femur, sacrum tumor(giant cell tumor), pooled kidney and kidney tumor (renal cell carcinomaclear-cell type), pooled liver and liver tumor (neuroendocrinecarcinoma), pooled fetal liver, pooled lung, fetal pancreas, pancreas,parotid gland, parotid tumor (sebaceous lymphadenoma), retroperitonealand suprglottic soft tissue, spleen, fetal spleen, spleen tumor(malignant lymphoma), diseased spleen (idiopathic thrombocytopenicpurpura), parathyroid, thyroid, thymus, tonsil ureter tumor(transitional cell carcinoma), pooled adrenal gland and adrenal tumor(pheochromocytoma), pooled lymph node tumor (Hodgkin's disease andmetastatic adenocarcinoma), pooled neck and calf muscles, and pooledbladder. MONOTXN05 pINCY This normalized treated monocyte cell tissuelibrary was constructed from 1.03 million independent clones from amonocyte tissue library. Starting RNA was made from RNA isolated fromtreated monocytes from peripheral blood removed from a 42-year-oldfemale. The cells were treated with interleukin-10 (IL-10) andlipopolysaccharide (LPS). The library was normalized in two rounds usingconditions adapted from Soares et al., PNAS (1994) 91: 9228-9232 andBonaldo et al., Genome Research 6 (1996): 791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. NGANNOT01 PSPORT1 Library was constructed using RNA isolated fromtumorous neuroganglion tissue removed from a 9-year-old Caucasian maleduring a soft tissue excision of the chest wall. Pathology indicated aganglioneuroma. Family history included asthma. OVARDIR01 PCDNA2.1 Thisrandom primed library was constructed using RNA isolated from rightovary tissue removed from a 45-year-old Caucasian female during totalabdominal hysterectomy, bilateral salpingo-oophorectomy, vaginalsuspension and fixation, and incidental appendectomy. Pathologyindicated stromal hyperthecosis of the right and left ovaries. Pathologyfor the matched tumor tissue indicated a dermoid cyst (benign cysticteratoma) in the left ovary. Multiple (3) intramural leiomyomata wereidentified. The cervix showed squamous metaplasia. Patient historyincluded metrorrhagia, female stress incontinence, alopecia, depressivedisorder, pneumonia, normal delivery, and deficiency anemia. Familyhistory included benign hypertension, atherosclerotic coronary arterydisease, hyperlipidemia, and primary tuberculous complex. OVARTUE01PCDNA2.1 This 5′ biased random primed library was constructed using RNAisolated from left ovary tumor tissue removed from a 44-year-old female.Pathology indicated grade 4 (of 4) serous carcinoma replacing both theright and left ovaries forming solid mass cystic masses. Neoplasticdeposits were identified in para-ovarian soft tissue. PGANNOT03 pINCYLibrary was constructed using RNA isolated from paraganglionic tumortissue removed from the intra-abdominal region of a 46-year-oldCaucasian male during exploratory laparotomy. Pathology indicated abenign paraganglioma and was associated with a grade 2 renal cellcarcinoma, clear cell type, which did not penetrate the capsule.Surgical margins were negative for tumor. SCORNOT04 pINCY Library wasconstructed using RNA isolated from cervical spinal cord tissue removedfrom a 32-year-old Caucasian male who died from acute pulmonary edemaand bronchopneumonia, bilateral pleural and pericardial effusions, andmalignant lymphoma (natural killer cell type). Patient history includedprobable cytomegalovirus infection, hepatic congestion and steatosis,splenomegaly, hemorrhagic cystitis, thyroid hemorrhage, and Bell'spalsy. Surgeries included colonoscopy, large intestine biopsy,adenotonsillectomy, and nasopharyngeal endoscopy and biopsy; treatmentincluded radiation therapy. SEMVNOT03 pINCY Library was constructedusing RNA isolated from seminal vesicle tissue removed from a56-year-old male during a radical prostatectomy. Pathology for theassociated tumor tissue indicated adenocarcinoma (Gleason grade 3 + 3).SINTFEE01 pINCY This 5′ biased random primed library was constructedusing RNA isolated from small intestine tissue removed from a Caucasianmale fetus who died from fetal demise. SINTFET03 pINCY Library wasconstructed using RNA isolated from small intestine tissue removed froma Caucasian female fetus, who died at 20 weeks' gestation. SINTNOR01PCDNA2.1 This random primed library was constructed using RNA isolatedfrom small intestine tissue removed from a 31-year-old Caucasian femaleduring Roux-en-Y gastric bypass. Patient history included clinicalobesity. TESTNOT03 PBLUESCRIPT Library was constructed using RNAisolated from testicular tissue removed from a 37-year-old Caucasianmale, who died from liver disease. Patient history included cirrhosis,jaundice, and liver failure. THYRNOT03 pINCY Library was constructedusing RNA isolated from thyroid tissue removed from the left thyroid ofa 28-year-old Caucasian female during a complete thyroidectomy.Pathology indicated a small nodule of adenomatous hyperplasia present inthe left thyroid. Pathology for the associated tumor tissue indicateddominant follicular adenoma, forming a well-encapsulated mass in theleft thyroid. UCMCNOT02 pINCY Library was constructed using RNA isolatedfrom mononuclear cells obtained from the umbilical cord blood of nineindividuals. UTREDIT07 pINCY Library was constructed using RNA isolatedfrom diseased endometrial tissue removed from a female duringendometrial biopsy. Pathology indicated in phase endometrium withmissing beta 3, Type II defects.

TABLE 7 Program Description Reference Parameter Threshold ABI A programthat removes vector sequences and masks Applied Biosystems, FACTURAambiguous bases in nucleic acid sequences. Foster City, CA. ABI/ A FastData Finder useful in Applied Biosystems, Mismatch <50% PARACELcomparing and annotating amino Foster City, CA; FDF acid or nucleic acidsequences. Paracel Inc., Pasadena, CA. ABI A program that assemblesnucleic acid sequences. Applied Biosystems, AutoAssembler Foster City,CA. BLAST A Basic Local Alignment Search Tool useful in Altschul, S.F.et al. (1990) ESTs: Probability sequence similarity search for aminoacid and nucleic J. Mol. Biol. 215: 403-410; value = 1.0E−8 acidsequences. BLAST includes five functions: Altschul, S.F. et al. (1997)or less; blastp, blastn, blastx, tblastn, and tblastx. Nucleic AcidsRes. 25: 3389-3402. Full Length sequences: Probability value = 1.0E−10or less FASTA A Pearson and Lipman algorithm that searches for Pearson,W. R. and ESTs: fasta E similarity between a query sequence and a groupof D. J. Lipman (1988) Proc. Natl. value = 1.06E−6; sequences of thesame type. FASTA comprises as Acad Sci. USA 85: 2444-2448; AssembledESTs: fasta least five functions: fasta, tfasta, fastx, tfastx, andPearson, W. R. (1990) Methods Enzymol. 183: 63-98; Identity = 95% orssearch. and Smith, T. F. and M. S. Waterman (1981) greater and Adv.Appl. Math. 2: 482-489. Match length = 200 bases or greater; fastx Evalue = 1.0E−8 or less; Full Length sequences: fastx score = 100 orgreater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S.and J. G. Henikoff (1991) Probability value = sequence against those inBLOCKS, PRINTS, Nucleic Acids Res. 19: 6565-6572; Henikoff, 1.0E−3 orless DOMO, PRODOM, and PFAM databases to search J. G. and S. Henikoff(1996) Methods for gene families, sequence homology, and structuralEnzymol. 266: 88-105; and Attwood, T. K. et fingerprint regions. al.(1997) J. Chem. Inf. Comput. Sci. 37: 417-424. HMMER An algorithm forsearching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol.PFAM, INCY, hidden Markov model (HMM)-based databases of 235: 1501-1531;Sonnhammer, E. L. L. et al. SMART or TIGRFAM protein family consensussequences, such as PFAM, (1988) Nucleic Acids Res. 26: 320-322; hits:Probability INCY, SMART and TIGRFAM. Durbin, R. et al. (1998) Our WorldView, in value = 1.0E−3 or less a Nutshell, Cambridge Univ. Press, pp.1-350. Signal peptide hits: Score = 0 or greater ProfileScan Analgorithm that searches for structural and Gribskov, M. et al. (1988)CABIOS 4: 61-66; Normalized quality sequence motifs in protein sequencesthat match Gribskov, M. et al. (1989) Methods score ≧ GCG sequencepatterns defined in Prosite. Enzymol. 183: 146-159; Bairoch, A. et al.specified “HIGH” (1997) Nucleic Acids Res. 25: 217-221. value for thatparticular Prosite motif. Generally, score = 1.4-2.1. Phred Abase-calling algorithm that examines automated Ewing, B. et al. (1998)Genome Res. 8: 175-185; sequencer traces with high sensitivity andprobability. Ewing, B. and P. Green (1998) Genome Res. 8: 186-194. PhrapA Phils Revised Assembly Program including Smith, T. F. and M. S.Waterman (1981) Adv. Score = 120 or greater; SWAT and CrossMatch,programs based on efficient Appl. Math. 2: 482-489; Smith, T. F. andMatch length = implementation of the Smith-Waterman algorithm, M. S.Waterman (1981) J. Mol. Biol. 147: 195-197; 56 or greater useful insearching sequence homology and and Green, P., University of assemblingDNA sequences. Washington, Seattle, WA. Consed A graphical tool forviewing and editing Phrap Gordon, D. et al. (1998) Genome Res. 8:195-202. assemblies. SPScan A weight matrix analysis program that scansprotein Nielson, H. et al. (1997) Protein Engineering Score = 3.5 orgreater sequences for the presence of secretory signal 10: 1-6;Claverie, J. M. and S. Audic (1997) peptides. CABIOS 12: 431-439. TMAP Aprogram that uses weight matrices to delineate Persson, B. and P. Argos(1994) J. Mol. Biol. transmembrane segments on protein sequences and237: 182-192; Persson, B. and P. Argos determine orientation. (1996)Protein Sci. 5: 363-371. TMHMMER A program that uses a hidden Markovmodel (HMM) Sonnhammer, E.L. et al. (1998) Proc. Sixth to delineatetransmembrane segments on protein Intl. Conf. on Intelligent Systems forMol. sequences and determine orientation. Biol., Glasgow et al., eds.,The Am. Assoc. for Artificial Intelligence (AAAI) Press, Menlo Park, CA,and MIT Press, Cambridge, MA, pp. 175-182. Motifs A program thatsearches amino acid sequences for Bairoch, A. et al. (1997) NucleicAcids Res. patterns that matched those defined in Prosite. 25: 217-221;Wisconsin Package Program Manual, version 9, page M51-59, GeneticsComputer Group, Madison, WI.

TABLE 8 SEQ Al- Al- Caucasian African Asian Hispanic ID EST CB1 EST lelelele Allele 1 Allele 1 Allele 1 Allele 1 NO: PID EST ID SNP ID SNP SNPAllele 1 2 Amino Acid frequency frequency frequency Frequency 94 75115303218974H1 SNP00049492 34 78 G G A M1 n/a n/a n/a n/a 94 75115304515573H1 SNP00149596 123 212 T C T I46 n/a n/a n/a n/a 95 75115352812434H1 SNP00049596 182 292 C C T L73 n/a n/a n/a n/a 95 75115353218974H1 SNP00049492 34 78 G G A M1 n/a n/a n/a n/a 96 75115362812434H1 SNP00149596 182 310 C C T L73 n/a n/a n/a n/a 96 75115363218974H1 SNP00049492 34 96 G G A M1 n/a n/a n/a n/a 97 75115831224254H1 SNP00144336 16 70 T T C V15 n/a n/a n/a n/a 97 75115831296182H1 SNP00095646 72 281 C C T C85 n/a n/a n/a n/a 97 75115831401267F6 SNP00069629 266 1190 C T C noncoding n/a n/a n/a n/a 977511583 157722F1 SNP00069628 327 976 T T C noncoding n/a n/a n/a n/a 977511583 1616725T6 SNP00059171 166 963 G T G noncoding n/a n/a n/a n/a 977511583 1616725T6 SNP00059172 138 991 C C T noncoding n/a n/a n/a n/a 977511583 1757780H1 SNP00007835 178 422 G G A L132 0.76 0.76 0.99 0.84 977511583 1757780H1 SNP00144337 127 371 G G A S115 n/a n/a n/a n/a 977511583 5095527F6 SNP00152200 374 422 A G A L132 n/a n/a n/a n/a 987511395 1630029H1 SNP00003610 131 806 G C G L268 0.13 n/a n/a n/a 987511395 1633719F6 SNP00023566 202 938 T C T F312 n/a n/a n/a n/a 997511647 1286725H1 SNP00010241 142 1169 G G A noncoding n/a n/a n/a n/a99 7511647 1286725T6 SNP00010241 73 1187 G G A noncoding n/a n/a n/a n/a99 7511647 2242360F6 SNP00010241 110 1201 A G A noncoding n/a n/a n/an/a 99 7511647 2242360T6 SNP00010241 41 1205 A G A noncoding n/a n/a n/an/a 99 7511647 2595325T6 SNP00010241 85 1175 A G A noncoding n/a n/a n/an/a 99 7511647 5021938T1 SNP00010241 78 1171 G G A noncoding n/a n/a n/an/a 99 7511647 6022930H1 SNP00128089 30 118 C C T R39 n/a n/a n/a n/a100 7510335 1212125H1 SNP00140490 174 2243 C C T noncoding n/a n/a n/an/a 100 7510335 1216827H1 SNP00150092 184 2325 C C T noncoding n/a n/an/a n/a 100 7510335 1291887H1 SNP00128337 147 1933 C C T noncoding n/an/a n/a n/a 100 7510335 1398850H1 SNP00060257 217 2108 C C T noncodingn/d n/d n/d n/d 100 7510335 1419179H1 SNP00060256 119 2001 C C Tnoncoding n/d n/a n/a n/a 100 7510335 1540254H1 SNP00033095 171 1589 C CT noncoding n/d n/d n/d n/d 100 7510335 1544766H1 SNP00147917 40 1076 TT C noncoding n/a n/a n/a n/a 100 7510335 1710273H1 SNP00147918 67 1498G G A noncoding n/a n/a n/a n/a 100 7510335 1804935H1 SNP00135525 201706 G G C noncoding n/a n/a n/a n/a 100 7510335 1961191H1 SNP00033095186 1590 C C T noncoding n/d n/d n/d n/d 100 7510335 2212721H1SNP00068498 134 579 G G C G154 n/a n/a n/a n/a 100 7510335 2212721H1SNP00146716 43 488 T C T D123 n/a n/a n/a n/a 100 7510335 223647H1SNP00060257 104 2107 C C T noncoding n/d n/d n/d n/d 100 75103352811126H1 SNP00033095 51 1587 C C T noncoding n/d n/d n/d n/d 1007510335 2961433H1 SNP00128337 126 1930 C C T noncoding n/a n/a n/a n/a100 7510335 3023579H1 SNP00128337 169 1932 C C T noncoding n/a n/a n/an/a 100 7510335 3090372H1 SNP00033095 208 1588 C C T noncoding n/d n/dn/d n/d 100 7510335 3106751H1 SNP00128337 136 1928 C C T noncoding n/an/a n/a n/a 100 7510335 3111223H1 SNP00147918 41 1497 G G A noncodingn/a n/a n/a n/a 100 7510335 3320948H1 SNP00147918 95 1496 G G Anoncoding n/a n/a n/a n/a 100 7510335 3497717H1 SNP00060256 169 2000 C CT noncoding n/d n/a n/a n/a 100 7510335 3534331H1 SNP00147917 69 1074 TT C noncoding n/a n/a n/a n/a 100 7510335 3604157H1 SNP00060257 222 2105C C T noncoding n/d n/d n/d n/d 100 7510335 3605357H1 SNP00060256 1141998 C C T noncoding n/d n/a n/a n/a 100 7510335 3674561H1 SNP00147918101 1489 A G A noncoding n/a n/a n/a n/a 100 7510335 3806218H1SNP00033095 135 1574 C C T noncoding n/d n/d n/d n/d 100 75103353946457H1 SNP00068498 169 577 G G C G153 n/a n/a n/a n/a 100 75103353946457H1 SNP00146716 78 486 C C T H123 n/a n/a n/a n/a 100 75103354042248H1 SNP00128538 27 1219 C C T noncoding n/a n/a n/a n/a 1007510335 4070502H1 SNP00060257 271 2106 C C T noncoding n/d n/d n/d n/d100 7510335 4070502H1 SNP00128337 96 1931 C C T noncoding n/a n/a n/an/a 100 7510335 4095392H1 SNP00140490 230 2240 C C T noncoding n/a n/an/a n/a 100 7510335 4118647H1 SNP00060256 26 1953 C C T noncoding n/dn/a n/a n/a 100 7510335 4125450H1 SNP00128538 196 1214 C C T noncodingn/a n/a n/a n/a 100 7510335 4516130H1 SNP00128538 153 1222 C C Tnoncoding n/a n/a n/a n/a 100 7510335 4668664H1 SNP00147918 175 1491 G GA noncoding n/a n/a n/a n/a 100 7510335 4776052H1 SNP00135525 9 1704 G GC noncoding n/a n/a n/a n/a 100 7510335 4838066H1 SNP00128337 143 1929 CC T noncoding n/a n/a n/a n/a 100 7510335 4850641H1 SNP00147917 5 1075 TT C noncoding n/a n/a n/a n/a 100 7510335 5025486H1 SNP00135525 47 1700G G C noncoding n/a n/a n/a n/a 100 7510335 5218718H1 SNP00140490 1032183 C C T noncoding n/a n/a n/a n/a 100 7510335 5802821H1 SNP00128337121 1898 C C T noncoding n/a n/a n/a n/a 100 7510335 5810857H1SNP00068498 158 576 G G C V153 n/a n/a n/a n/a 100 7510335 5971080H1SNP00150092 320 425 T C T L102 n/a n/a n/a n/a 100 7510335 5987440H1SNP00150092 47 2324 C C T noncoding n/a n/a n/a n/a 100 75103356164432H1 SNP00033095 155 1583 C C T noncoding n/d n/d n/d n/d 1007510335 6217485H1 SNP00128337 402 1834 C C T noncoding n/a n/a n/a n/a100 7510335 6243311H1 SNP00150091 88 799 C C T S227 n/a n/a n/a n/a 1007510335 6251127H1 SNP00033095 264 1552 C C T noncoding n/d n/d n/d n/d100 7510335 6362371H1 SNP00135525 320 1779 G G C noncoding n/a n/a n/an/a 100 7510335 6472431H1 SNP00128337 152 1812 C C T noncoding n/a n/an/a n/a 100 7510335 683181H1 SNP00146716 48 487 C C T A123 n/a n/a n/an/a 100 7510335 687860H1 SNP00135525 62 1716 G G C noncoding n/a n/a n/an/a 100 7510335 7048680H1 SNP00147916 97 859 G G A R247 n/a n/a n/a n/a100 7510335 837712H1 SNP00147917 8 1073 T T C noncoding n/a n/a n/a n/a101 7510337 1212125H1 SNP00140490 174 2220 C C T noncoding n/a n/a n/an/a 101 7510337 1216827H1 SNP00150092 184 2302 C C T noncoding n/a n/an/a n/a 101 7510337 1291887H1 SNP00128337 147 1838 C C T I573 n/a n/an/a n/a 101 7510337 1459431H1 SNP00060256 53 1906 C C T A596 n/d n/a n/an/a 101 7510337 1540254H1 SNP00033095 171 1494 C C T R459 n/d n/d n/dn/d 101 7510337 1544766H1 SNP00147917 40 981 T T C F288 n/a n/a n/a n/a101 7510337 1710273H1 SNP00147918 67 1403 G G A K428 n/a n/a n/a n/a 1017510337 1804935H1 SNP00135525 20 1611 G G C A498 n/a n/a n/a n/a 1017510337 1961191H1 SNP00033095 186 1495 C C T P459 n/d n/d n/d n/d 1017510337 1964030H1 SNP00060257 187 2015 C C T noncoding n/d n/d n/d n/d101 7510337 2212721H1 SNP00068498 134 579 G G C G154 n/a n/a n/a n/a 1017510337 2212721H1 SNP00146716 43 488 T C T D123 n/a n/a n/a n/a 1017510337 2811126H1 SNP00033095 51 1492 C C T S458 n/d n/d n/d n/d 1017510337 3023579H1 SNP00128337 169 1837 C C T T573 n/a n/a n/a n/a 1017510337 3090372H1 SNP00033095 208 1493 C C T F458 n/d n/d n/d n/d 1017510337 3111223H1 SNP00147918 41 1402 G G A R428 n/a n/a n/a n/a 1017510337 3320948H1 SNP00147918 95 1401 G G A E428 n/a n/a n/a n/a 1017510337 3534331H1 SNP00147917 69 979 T T C V287 n/a n/a n/a n/a 1017510337 3574410H1 SNP00128337 184 1835 C C T A572 n/a n/a n/a n/a 1017510337 3674561H1 SNP00147918 101 1394 A G A A425 n/a n/a n/a n/a 1017510337 3806218H1 SNP00033095 135 1479 C C T H454 n/d n/d n/d n/d 1017510337 3946457H1 SNP00068498 169 577 G G C G153 n/a n/a n/a n/a 1017510337 3946457H1 SNP00146716 78 486 C C T H123 n/a n/a n/a n/a 1017510337 4042248H1 SNP00128538 27 1124 C C T H335 n/a n/a n/a n/a 1017510337 4070502H1 SNP00060257 271 2083 C C T noncoding n/d n/d n/d n/d101 7510337 4118647H1 SNP00060256 26 1858 C C T A580 n/d n/a n/a n/a 1017510337 4125450H1 SNP00128538 196 1119 C C T L334 n/a n/a n/a n/a 1017510337 4277305H1 SNP00128337 158 1836 C C T L573 n/a n/a n/a n/a 1017510337 4516130H1 SNP00128538 153 1127 C C T I336 n/a n/a n/a n/a 1017510337 4668664H1 SNP00147918 175 1396 G G A G426 n/a n/a n/a n/a 1017510337 4776052H1 SNP00135525 9 1609 G G C S497 n/a n/a n/a n/a 1017510337 4838066H1 SNP00128337 143 1834 C C T A572 n/a n/a n/a n/a 1017510337 4850641H1 SNP00147917 5 980 T T C G287 n/a n/a n/a n/a 1017510337 5025486H1 SNP00135525 47 1605 G G C G496 n/a n/a n/a n/a 1017510337 5218718H1 SNP00140490 103 2160 C C T noncoding n/a n/a n/a n/a101 7510337 5596417H1 SNP00150091 92 794 C C T A225 n/a n/a n/a n/a 1017510337 5802821H1 SNP00128337 121 1803 C C T Q562 n/a n/a n/a n/a 1017510337 5810857H1 SNP00068498 158 576 G G C V153 n/a n/a n/a n/a 1017510337 5971080H1 SNP00150092 320 425 T C T L102 n/a n/a n/a n/a 1017510337 5987440H1 SNP00150092 47 2301 C C T noncoding n/a n/a n/a n/a101 7510337 6164432H1 SNP00033095 155 1488 C C T L457 n/d n/d n/d n/d101 7510337 6217485H1 SNP00128337 402 1739 C C T L540 n/a n/a n/a n/a101 7510337 6243311H1 SNP00150091 88 799 C C T S227 n/a n/a n/a n/a 1017510337 6251127H1 SNP00033095 264 1457 C C T P446 n/d n/d n/d n/d 1017510337 6362371H1 SNP00135525 320 1684 G G C S522 n/a n/a n/a n/a 1017510337 6472431H1 SNP00128337 152 1717 C C T A533 n/a n/a n/a n/a 1017510337 6501461H1 SNP00060257 461 2085 C C T noncoding n/d n/d n/d n/d101 7510337 6802209J1 SNP00060257 231 2014 C C T noncoding n/d n/d n/dn/d 101 7510337 683181H1 SNP00146716 48 487 C C T A123 n/a n/a n/a n/a101 7510337 687860H1 SNP00135525 62 1621 G G C R501 n/a n/a n/a n/a 1017510337 7048680H1 SNP00147916 97 859 G G A R247 n/a n/a n/a n/a 1017510337 837712H1 SNP00147917 8 978 T T C C287 n/a n/a n/a n/a 1027510353 1420447H1 SNP00147377 42 525 C C T T171 n/a n/a n/a n/a 1027510353 1493080H1 SNP00149399 154 225 A A G Q71 n/a n/a n/a n/a 1027510353 2314923H1 SNP00147378 248 576 T T C L188 n/a n/a n/a n/a 1027510353 2569281H1 SNP00149762 219 595 C C T A194 n/a n/a n/a n/a 1027510353 2848514H1 SNP00149399 135 222 A A G D70 n/a n/a n/a n/a 1027510353 3593344H1 SNP00149399 23 223 A A G E70 n/a n/a n/a n/a 1027510353 4187759H1 SNP00099615 27 650 T T G C213 n/d n/a n/a n/a 1027510353 4201932H1 SNP00099615 26 648 T T G F212 n/d n/a n/a n/a 1027510353 4640886H1 SNP00147377 205 524 C C T L171 n/a n/a n/a n/a 1027510353 5583090H1 SNP00149399 149 224 A A G K71 n/a n/a n/a n/a 1027510353 5895839H1 SNP00099615 249 647 T T G Y212 n/d n/a n/a n/a 1027510353 5895839H1 SNP00149762 194 592 C C T D193 n/a n/a n/a n/a 1027510353 6567150H1 SNP00092265 520 1209 T T C V399 n/d n/a n/a n/a 1037510470 1417623H1 SNP00037122 190 1726 T T C noncoding n/a n/a n/a n/a103 7510470 217091H1 SNP00009165 27 1912 G G A noncoding n/a n/a n/a n/a103 7510470 2364930H1 SNP00154397 130 1829 G G C noncoding n/a n/a n/an/a 103 7510470 2367975H1 SNP00122563 54 1833 C C T noncoding n/d n/an/a n/a 103 7510470 2371106H1 SNP00122563 186 1848 C C T noncoding n/dn/a n/a n/a 103 7510470 2562140H1 SNP00126019 119 144 G A G R44 n/a n/an/a n/a 103 7510470 2562140H1 SNP00126020 275 300 A A G D96 n/a n/a n/an/a 103 7510470 2647388H1 SNP00154397 14 1828 G G C noncoding n/a n/an/a n/a 103 7510470 2659667H1 SNP00037122 54 1725 T T C noncoding n/an/a n/a n/a 103 7510470 2659667H1 SNP00122563 176 1847 C C T noncodingn/d n/a n/a n/a 103 7510470 2664626H1 SNP00126021 147 303 T T C V97 n/an/a n/a n/a 103 7510470 2664980H1 SNP00058384 165 1234 A A C R407 n/an/a n/a n/a 103 7510470 2958538H1 SNP00075517 240 259 C T C D82 0.44 n/an/a n/a 103 7510470 2960825H1 SNP00037122 73 1716 T T C noncoding n/an/a n/a n/a 103 7510470 3501789H1 SNP00126019 129 143 G A G G44 n/a n/an/a n/a 103 7510470 3502578H1 SNP00126020 259 299 A A G N96 n/a n/a n/an/a 103 7510470 3502578H1 SNP00126021 262 302 T T C L97 n/a n/a n/a n/a103 7510470 7011485H1 SNP00075517 66 252 T T C M80 0.44 n/a n/a n/a 1037510470 7012255H1 SNP00106403 397 1246 A A G S411 n/d n/a n/a n/a 1037510470 7014056H1 SNP00058383 61 873 T C T I287 n/d n/a n/a n/a 1037510470 7014228H1 SNP00075518 135 1444 T C T R477 n/a n/a n/a n/a 1037510470 7014873H1 SNP00037123 487 2110 G G A noncoding n/a n/a n/a n/a103 7510470 7371634H1 SNP00126022 485 502 A G A A163 n/a n/a n/a n/a 1037510470 7650627H1 SNP00119673 192 1277 G G A A422 n/d n/a n/a n/a 1037510470 940290H1 SNP00154397 117 1827 G G C noncoding n/a n/a n/a n/a104 7504648 1212125H1 SNP00140490 174 2054 C C T noncoding n/a n/a n/an/a 104 7504648 1216827H1 SNP00150092 184 2136 C C T noncoding n/a n/an/a n/a 104 7504648 1291887H1 SNP00128337 147 1744 C C T noncoding n/an/a n/a n/a 104 7504648 1398850H1 SNP00060257 217 1919 C C T noncodingn/d n/d n/d n/d 104 7504648 1419179H1 SNP00060256 119 1812 C C Tnoncoding n/d n/a n/a n/a 104 7504648 1540254H1 SNP00033095 171 1498 C CT R459 n/d n/d n/d n/d 104 7504648 1544766H1 SNP00147917 40 985 T T CF288 n/a n/a n/a n/a 104 7504648 1710273H1 SNP00147918 67 1407 G G AK428 n/a n/a n/a n/a 104 7504648 1961191H1 SNP00033095 186 1499 C C TP459 n/d n/d n/d n/d 104 7504648 2212721H1 SNP00068498 134 583 G G CG154 n/a n/a n/a n/a 104 7504648 2212721H1 SNP00146716 43 492 T C T D123n/a n/a n/a n/a 104 7504648 223647H1 SNP00060257 104 1918 C C Tnoncoding n/d n/d n/d n/d 104 7504648 2811126H1 SNP00033095 51 1496 C CT S458 n/d n/d n/d n/d 104 7504648 2961433H1 SNP00128337 126 1741 C C Tnoncoding n/a n/a n/a n/a 104 7504648 3023579H1 SNP00128337 169 1743 C CT noncoding n/a n/a n/a n/a 104 7504648 3089579H1 SNP00128337 210 1742 CC T noncoding n/a n/a n/a n/a 104 7504648 3090372H1 SNP00033095 208 1497C C T F458 n/d n/d n/d n/d 104 7504648 3106751H1 SNP00128337 136 1739 CC T noncoding n/a n/a n/a n/a 104 7504648 3111223H1 SNP00147918 41 1406G G A R428 n/a n/a n/a n/a 104 7504648 3320948H1 SNP00147918 95 1405 G GA E428 n/a n/a n/a n/a 104 7504648 3497717H1 SNP00060256 169 1811 C C Tnoncoding n/d n/a n/a n/a 104 7504648 3534331H1 SNP00147917 69 983 T T CV287 n/a n/a n/a n/a 104 7504648 3604157H1 SNP00060257 222 1916 C C Tnoncoding n/d n/d n/d n/d 104 7504648 3605357H1 SNP00060256 114 1809 C CT noncoding n/d n/a n/a n/a 104 7504648 3674561H1 SNP00147918 101 1398 AG A A425 n/a n/a n/a n/a 104 7504648 3806218H1 SNP00033095 135 1483 C CT H454 n/d n/d n/d n/d 104 7504648 3946457H1 SNP00068498 169 581 G G CG153 n/a n/a n/a n/a 104 7504648 3946457H1 SNP00146716 78 490 C C T H123n/a n/a n/a n/a 104 7504648 4042248H1 SNP00128538 27 1128 C C T H335 n/an/a n/a n/a 104 7504648 4070502H1 SNP00060257 271 1917 C C T noncodingn/d n/d n/d n/d 104 7504648 4095392H1 SNP00140490 230 2051 C C Tnoncoding n/a n/a n/a n/a 104 7504648 4118647H1 SNP00060256 26 1764 C CT noncoding n/d n/a n/a n/a 104 7504648 4125450H1 SNP00128538 196 1123 CC T L334 n/a n/a n/a n/a 104 7504648 4516130H1 SNP00128538 153 1131 C CT I336 n/a n/a n/a n/a 104 7504648 4668664H1 SNP00147918 175 1400 G G AG426 n/a n/a n/a n/a 104 7504648 4838066H1 SNP00128337 143 1740 C C Tnoncoding n/a n/a n/a n/a 104 7504648 4850641H1 SNP00147917 5 984 T T CG287 n/a n/a n/a n/a 104 7504648 5218718H1 SNP00140490 103 1994 C C Tnoncoding n/a n/a n/a n/a 104 7504648 5596417H1 SNP00150091 92 798 C C TA225 n/a n/a n/a n/a 104 7504648 5802821H1 SNP00128337 121 1709 C C Tnoncoding n/a n/a n/a n/a 104 7504648 5810857H1 SNP00068498 158 580 G GC V153 n/a n/a n/a n/a 104 7504648 5971080H1 SNP00150092 320 429 T C TL102 n/a n/a n/a n/a 104 7504648 5987440H1 SNP00150092 47 2135 C C Tnoncoding n/a n/a n/a n/a 104 7504648 6164432H1 SNP00033095 155 1492 C CT L457 n/d n/d n/d n/d 104 7504648 6243311H1 SNP00150091 88 803 C C TS227 n/a n/a n/a n/a 104 7504648 6472431H1 SNP00060257 327 1787 C C Tnoncoding n/d n/d n/d n/d 104 7504648 6472431H1 SNP00128337 152 1612 C CT Q497 n/a n/a n/a n/a 104 7504648 683181H1 SNP00146716 48 491 C C TA123 n/a n/a n/a n/a 104 7504648 7048680H1 SNP00147916 97 863 G G A R247n/a n/a n/a n/a 104 7504648 837712H1 SNP00147917 8 982 T T C C287 n/an/a n/a n/a 105 7512747 1215521H1 SNP00096877 235 378 G G C M104 n/a n/an/a n/a 105 7512747 1215521H1 SNP00134446 201 344 A A G Q93 n/a n/a n/an/a 105 7512747 2060954R6 SNP00096877 415 377 G G C R104 n/a n/a n/a n/a105 7512747 2060954R6 SNP00134446 381 343 A A G K93 n/a n/a n/a n/a 1057512747 7754178J1 SNP00096877 358 355 G G C A97 n/a n/a n/a n/a 1057512747 7754178J1 SNP00134446 324 321 A A G R85 n/a n/a n/a n/a 1067510146 1417623H1 SNP00037122 190 2017 T T C noncoding n/a n/a n/a n/a106 7510146 217091H1 SNP00009165 27 2203 G G A noncoding n/a n/a n/a n/a106 7510146 2364930H1 SNP00154397 130 2120 G G C noncoding n/a n/a n/an/a 106 7510146 2367975H1 SNP00122563 54 2139 C C T noncoding n/d n/an/a n/a 106 7510146 2562140H1 SNP00126019 119 142 G A G R44 n/a n/a n/an/a 106 7510146 2562140H1 SNP00126020 275 298 A A G D96 n/a n/a n/a n/a106 7510146 2564755H1 SNP00058384 80 1525 A A C noncoding n/a n/a n/an/a 106 7510146 2664626H1 SNP00126021 147 301 T T C V97 n/a n/a n/a n/a106 7510146 2958538H1 SNP00075517 240 257 C T C D82 0.44 n/a n/a n/a 1067510146 2962264T6 SNP00009165 179 2222 G G A noncoding n/a n/a n/a n/a106 7510146 2962264T6 SNP00037122 365 2036 C T C noncoding n/a n/a n/an/a 106 7510146 2962264T6 SNP00122563 243 2158 C C T noncoding n/d n/an/a n/a 106 7510146 7012255H1 SNP00106403 397 1537 A A G noncoding n/dn/a n/a n/a 106 7510146 7013451F8 SNP00037123 382 2401 G G A noncodingn/a n/a n/a n/a 106 7510146 7014056H1 SNP00058383 61 871 T C T I287 n/dn/a n/a n/a 106 7510146 7014228H1 SNP00075518 135 1735 T C T noncodingn/a n/a n/a n/a 106 7510146 7370025H1 SNP00058384 343 1526 A A Cnoncoding n/a n/a n/a n/a 106 7510146 7371634H1 SNP00126019 127 151 G AG S47 n/a n/a n/a n/a 106 7510146 7371634H1 SNP00126020 283 307 A A GK99 n/a n/a n/a n/a 106 7510146 7371634H1 SNP00126021 286 310 T T C L100n/a n/a n/a n/a 106 7510146 7371634H1 SNP00126022 485 510 A G A N167 n/an/a n/a n/a 106 7510146 7650307J2 SNP00058383 557 873 C C T R288 n/d n/an/a n/a 106 7510146 7651139H1 SNP00126022 452 500 A G A A163 n/a n/a n/an/a 106 7510146 7652407H2 SNP00009165 298 2202 G G A noncoding n/a n/an/a n/a 106 7510146 7652407H2 SNP00037122 484 2016 T T C noncoding n/an/a n/a n/a 106 7510146 7652407H2 SNP00122563 362 2138 C C T noncodingn/d n/a n/a n/a

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:8-9, SEQ ID NO:11-13, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:29-34,SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:46, and SEQ IDNO:50, c) a polypeptide comprising a naturally occurring amino acidsequence at least 95% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:4 and SEQ ID NO:23, d) a polypeptidecomprising a naturally occurring amino acid sequence at least 98%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:10 and SEQ ID NO:35, e) a polypeptide comprising anaturally occurring amino acid sequence at least 94% identical to theamino acid sequence of SEQ ID NO:17, f) a polypeptide comprising anaturally occurring amino acid sequence at least 97% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:37and SEQ ID NO:53, g) a polypeptide comprising a naturally occurringamino acid sequence at least 93% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:39 and SEQ ID NO:49, h)a polypeptide comprising a naturally occurring amino acid sequence atleast 91% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:47 and SEQ ID NO:51 i) a polypeptide consistingessentially of a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42-43, SEQ ID NO:45, SEQ IDNO:48, and SEQ ID NO:52, j) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53, and k) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-53.
 2. An isolated polypeptide of claim 1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-53.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:54-106.
 6. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. (canceled)
 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-53.
 11. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 12. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:54-106, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:54-57, SEQ ID NO:60-70, SEQ ID NO:73-89, SEQ ID NO:91-93, SEQ IDNO:97, and SEQ ID NO:106, c) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 98% identical to thepolynucleotide sequence of SEQ ID NO:58, d) a polynucleotide comprisinga naturally occurring polynucleotide sequence at least 92% identical tothe polynucleotide sequence of SEQ ID NO:71, e) a polynucleotidecomprising a naturally occurring polynucleotide sequence at least 91%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:72 and SEQ ID NO:90, f) a polynucleotidecomprising a naturally occurring polynucleotide sequence at least 93%identical to the polynucleotide sequence of SEQ ID NO:102, g) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 96% identical to the polynucleotide sequence of SEQ ID NO:100,h) a polynucleotide comprising a naturally occurring polynucleotidesequence at least 97% identical to a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:101 and SEQ ID NO:103, i) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 99% identical to the polynucleotide sequence of SEQ ID NO:104,j) a polynucleotide consisting essentially of a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence selected from the group consisting of SEQ ID NO:94-96, SEQ IDNO:98-99, and SEQ ID NO:105, k) a polynucleotide complementary to apolynucleotide of a), l) a polynucleotide complementary to apolynucleotide of b), m) a polynucleotide complementary to apolynucleotide of c), n) a polynucleotide complementary to apolynucleotide of d), o) a polynucleotide complementary to apolynucleotide of e), p) a polynucleotide complementary to apolynucleotide of f), q) a polynucleotide complementary to apolynucleotide of g), r) a polynucleotide complementary to apolynucleotide of h), s) a polynucleotide complementary to apolynucleotide of i), t) a polynucleotide complementary to apolynucleotide of j), and u) an RNA equivalent of a)-t).
 13. (canceled)14. A method of detecting a target polynucleotide in a sample, saidtarget polynucleotide having a sequence of a polynucleotide of claim 12,the method comprising: a) hybridizing the sample with a probe comprisingat least 20 contiguous nucleotides comprising a sequence complementaryto said target polynucleotide in the sample, and which probespecifically hybridizes to said target polynucleotide, under conditionswhereby a hybridization complex is formed between said probe and saidtarget polynucleotide or fragments thereof, and b) detecting thepresence or absence of said hybridization complex, and, optionally, ifpresent, the amount thereof.
 15. (canceled)
 16. A method of detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)amplifying said target polynucleotide or fragment thereof usingpolymerase chain reaction amplification, and b) detecting the presenceor absence of said amplified target polynucleotide or fragment thereof,and, optionally, if present, the amount thereof.
 17. A compositioncomprising a polypeptide of claim 1 and a pharmaceutically acceptableexcipient.
 18. A composition of claim 17, wherein the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:1-53.
 19. (canceled)
 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample. 21.(canceled)
 22. (canceled)
 23. A method of screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample. 24.(canceled)
 25. (canceled)
 26. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, the method comprising:a) combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 27. (canceled)
 28. Amethod of screening a compound for effectiveness in altering expressionof a target polynucleotide, wherein said target polynucleotide comprisesa sequence of claim 5, the method comprising: a) exposing a samplecomprising the target polynucleotide to a compound, under conditionssuitable for the expression of the target polynucleotide, b) detectingaltered expression of the target polynucleotide, and c) comparing theexpression of the target polynucleotide in the presence of varyingamounts of the compound and in the absence of the compound.
 29. A methodof assessing toxicity of a test compound, the method comprising: a)treating a biological sample containing nucleic acids with the testcompound, b) hybridizing the nucleic acids of the treated biologicalsample with a probe comprising at least 20 contiguous nucleotides of apolynucleotide of claim 12 under conditions whereby a specifichybridization complex is formed between said probe and a targetpolynucleotide in the biological sample, said target polynucleotidecomprising a polynucleotide sequence of a polynucleotide of claim 12 orfragment thereof, c) quantifying the amount of hybridization complex,and d) comparing the amount of hybridization complex in the treatedbiological sample with the amount of hybridization complex in anuntreated biological sample, wherein a difference in the amount ofhybridization complex in the treated biological sample is indicative oftoxicity of the test compound. 30-161. (canceled)